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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Weight of evidence. Read-across approach. Experimental results from reproductive toxicity studies performed with some analogues are available:

Diisodecyl phthalate (DIDP): 2 two-generation reproductive toxicity studies in accordance with EU Method B.35 and EPA guideline OPPTS 870.3800. GLP study. Based on read across approach, DUP is determined to have a NOAEL of 0.85% in diet (approximately 637.69 mg/kg/day) for fertility, reproduction function and developmental landmarks and 0.064% in diet (approximately 53.14 mg/kg/day) for F2 offspring survival.

Diisononyl phthalate (DINP): two-generation reproductive toxicity study in accordance with EU method B.35 and EPA guideline OTS 798.4700. GLP study. Based on read across approach, DUP is estimated to have a NOAEL for reproductive performance of 0.91% in the diet (approximately 567.02 mg/kg/day).

Di-(C9-C11 alkyl) phthalate (D911P): two-generation reproductive toxicity study in accordance with EPA guideline OPPTS 870.3800. GLP study. Based on read across approach, DUP is considered without adverse effects upon fertility or reproductive performance in rats at dietary levels that induce systemic toxicity and the NOAEL is determined to be 0.53% in the diet.

In a weight of evidence approach and after applying read-across with the results of these studies with different phthalates, DUP can be considered without adverse effects upon fertility or reproductive performance up to the highest concentrations tested in each study. In one study a significant reduction in F2 offspring survival was found. However, taking into account that this effect was not found in the other 2 studies with other phthalates and also that no evidence of teratogenic effects was observed in a pre-natal developmental toxicity performed on DUP, it was not considered relevant enough to justify a classification of the test susbstance.

Link to relevant study records

Referenceopen allclose all

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
EU Method B.35 (Two-Generation Reproduction Toxicity Test)
Version / remarks:
EC Dangerous Substances Directive (67/548/EEC), Annex V, Part B
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:
not specified
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Crl:CD BR - VAF/PLUS®
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Stone Ridge, New York (males) and Portage, Michigan (females). In study B male rats were purchased from the Raleigh Facility, Raleigh, North Carolina.
- Age at study initiation: Not reported
- Weight at study initiation: not reported
- Housing: Animals were individually housed in suspended stainless steel and wire mesh cages.
- Diet (e.g. ad libitum): certified rodent chow (PMI Feeds, Inc., Richmond, IN), ad libitum.
- Water (e.g. ad libitum): automatic watering system, ad libitum.
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25ºC
- Humidity (%): 40-70 %
- Photoperiod: 12-h light/dark cycle

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly (stability data confirmed that DIDP was stable in feed at room temperature for at least 14 days)
- Mixing appropriate amounts with (Type of food): certified rodent chow (PMI Feeds, Inc., Richmond, IN).
- Storage temperature of food: Not reported

Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: The mating period ended when all females were confirmed mated or approx. 2 weeks had elapsed.
- Proof of pregnancy: copulatory plug or sperm in a vaginal rinse referred to as gestational day (GD) 0.
- Further matings after two unsuccessful attempts: Not specified
- After successful mating each pregnant female was caged (how): After confirmation of mating, each mated female was returned to its own cage.
- Any other deviations from standard protocol: N/A
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test material was verified by gas chromatography and infrared analysis. Stability data confirmed that DIDP was stable in feed at room temperature for at least 14 days.
The actual concentrations used in the first study, based on dietary analysis, were 0.200 ± 0.010%, 0.401 ± 0.018%, and 0.807 ± 0.037%. In the second study the analytically confirmed concentrations were 0.019 ± 0.001%, 0.058 ± 0.002%, 0.192 ± 0.010% and 0.387 ± 0.019%.
Duration of treatment / exposure:
Males P1 and P2: 10 weeks prior to mating, through mating and until the day prior to euthanasia, following delivery of the last litter they sired.
Females P1 and P2: 10 weeks prior to mating, through mating, gestation and lactation and until sacrifice after weaning of offspring animals on PND 21.
Frequency of treatment:
constant exposure 7 days/week
Details on study schedule:
- Selection of parents from F1 generation when pups were 21 days of age.
Each litter was represented by at least one pup per sex if possible. Sibling matings were avoided.
Dose / conc.:
0 other: %
Remarks:
nominal in diet, study A and B
Dose / conc.:
0.02 other: %
Remarks:
Nominal in diet, study B
Dose / conc.:
0.06 other: %
Remarks:
nominal in diet, study B
Dose / conc.:
0.2 other: %
Remarks:
nominal in diet, study A and B
Dose / conc.:
0.4 other: %
Remarks:
nominal in diet, study A and B
Dose / conc.:
0.8 other: %
Remarks:
nominal in diet, study A
No. of animals per sex per dose:
P1 generation: 30
F1 generation: 30

Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale:
Dietary levels selected for the initial study A were based on results from a preliminary one-generation study. In this study, rats (10/sex/dose) were fed DIDP at dietary levels of 0.25, 0.50, 0.75, and 1.0% for approximately 10 weeks prior to mating and throughout the mating period. The males were sacrificed after mating, but females continued to be exposed throughout gestation and lactation, until weaning of the offspring on PND 21. Based on reductions in offspring and adult body weights at 0.75% and/or 1%, the dietary levels of DIDP selected for the initial two-generation study were 0.2, 0.4, and 0.8%.
Positive control:
N/A
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Males: weekly. Females: weekly, during gestation on GD 0, 7, 14, and 21, and during lactation on PND 0, 4, 7, 14, and 21.

BODY WEIGHT: Yes
- Time schedule for examinations: Males: weekly. Females: weekly, during gestation on GD 0, 7, 14, and 21, and during lactation on PND 0, 4, 7, 14, and 21..

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): yes. Parental food consumption was determined at the same time as the body weight measurements, except during mating.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

Oestrous cyclicity (parental animals):
The estrous cycle of each P1 and F1 female was evaluated daily by vaginal smears beginning three weeks prior to mating, continuing until the end of cohabitation, and on the day of sacrifice.
Sperm parameters (parental animals):
Parameters examined in P2 male generation:
Samples of sperm from the left distal caudal epididymis (or proximal vas deferens) were collected at scheduled terminal sacrifice from F2 male offspring and evaluated for the percentage of progressively motile sperm and sperm morphology. Also the entire left caudal epididymis was minced in saline to enumerate the total number of sperm (cauda reserves), and the left testis was homogenized to quantify the total number of homogenization resistant spermatids. Total cauda sperm counts and sperm motility were evaluated using the Hamilton-Thorne IVOS 2000 system for computer assisted sperm analysis (CASA).
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 4/sex/litter (as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
Litters from the P1 and F1 generations were examined twice daily for general appearance of the pups and dead offspring. All pups were counted, sexed, weighed, and examined externally on PND 0, 1, 4, 7, 14, and 21. Beginning on PND 29, all surviving F1 female offspring were examined daily for vaginal opening. These examinations continued until all animals reached criteria (i.e. vaginal opening).
In the Study B nipple retention and anogenital distance were measured, as were vaginal patency and preputial separation

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals following delivery of the last litter they sired.
- Maternal animals: All surviving animals after the last litter of each generation was weaned.

GROSS NECROPSY
- Complete gross postmortem examinations were conducted on all animals in the study.

HISTOPATHOLOGY / ORGAN WEIGHTS
Liver, kidneys (paired), testes (individual), prostate, seminal vesicles,right epididymis (total and cauda), ovaries (individual), uterus, and brain from all parental animals that survived to scheduled termination were removed and weighed. The pituitary, testes, epididymides, prostate, seminal vesicles, vagina, uterus, ovaries, mammary gland, oviducts, thymus, adrenals, coagulating gland, kidney, liver and gross lesions from all parental animals in the control and 0.8% dose groups were examined microscopically.
In addition, the reproductive organs of all animals in the 0.2% and 0.4% dose groups that had abnormal sperm, estrous cycles, or failed to produce viable litters were examined. The testes of the P1 and F1 males were preserved in Bouin's solution. All other tissues were fixed in 10% neutral formalin. The tissues were processed, embedded in paraffin, sectioned at 5 µm and stained with hematoxylin and eosin. Five sections of each ovary from females in the control and high dose were examined for oocyte evaluation.
In the Study B pathologic examinations were limited to the organs with microscopic findings in the first study (i.e. liver and kidney).
Postmortem examinations (offspring):
SACRIFICE
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
-Complete gross postmortem examinations were conducted on all animals in the study.
Statistics:
Quantitative continuous variables were analyzed for statistical significance. Bartlett's test of homogeneity of variance was used to determine if the groups had equal variance at the 1% level of significance. If the variances were equivalent, the groups were compared by using a standard one-way analysis of variance (ANOVA). If significant differences among the means were indicated, Dunnet's test was performed to determine which treated groups differed from control. In addition to the ANOVA, a standard regression analysis for linear response in the dose groups was performed. If the groups did not have equivalent variances at the 1% level, then a Kruskal-Wallis test (nonparametric) was used to assess differences in group means. If the means were different, Dunn's Rank Sum test (nonparametric) was used to determine which treatment groups differed significantly from control. In addition to the KW test, Jonckheere's test (nonparametric) for ordered response was also performed. Pup weight was analyzed by a mixed model of covariance with pups nested within dams, dams nested within dose, and total litter size as the covariant. If b.w. differences in groups were identified, the least squares means and the least significant difference test were used to determine which groups differed from the control group. Parental reproductive and offspring survival incidence data were evaluated by chi-square test analysis. Each treatment group was compared to the controls by using a 2x2 Fisher Exact test. Armitage's test for linear trend in the dosage groups was also performed.Sperm and oocyte count data were transformed by Blom’s transformation and analyzed by standard one way ANOVA. Residuals from the model were tested for normality by the Shapiro-Wilk test. Developmental landmarks were analyzed separately for each sex using a cumulative logistic regression model. Offspring survival was further assessed by fitting a modified probit model to the F2 offspring PND 0–4 survival data.
Reproductive indices:
Male mating index: no. males confirmed mated/no. males used for mating.
Male fertility index: no. males impregnating females/no. males used for mating.
Female fertility index: no. females confirmed mated/no. females paired.
Female fecundity index: no. females pregnant (excluding unconfirmed mated females)/no. females confirmed mated.
Gestational index: no. females with live litters/no. females pregnant.
Offspring viability indices:
Live birth index: no. live pups at birth/no. of pups born.
Day 1 survival index: no. live pups at Day 1/no. live pups at Day 0.
Day 4 survival index: no. live pups at Day 4/no. live pups at Day 0.
Day 7 survival index: no. live pups at Day 7/no. live pups at Day 4 (postcull).
Day 14 survival index: no. live pups at Day 14/no. live pups at Day 7.
Day 21 survival index: no. live pups at Day 21/no. live pups at Day 14.
Viability at weaning index: no. live pups at Day 21/no. live pups at Day 4 (postcull).
Clinical signs:
no effects observed
Description (incidence and severity):
Study A and B: There were no important or dose dependent clinical signs of toxicity in either the P1 males or females in any dose group.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
Study A: All but two parental (P1) animals survived to scheduled termination. Although the causes of death were not determined, both were from the control group so clearly did not die as a consequence of treatment.
Study B: there were no treatment-related effects on parental survival.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Study A: Male and female body weights in the 0.8% dose groups were significantly (ca. 9 to 10%) below control values during the premating period, as were female body weights during gestation and lactation.
Study B: there were no differences in body weights.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Study A and B: there were no differences in food consumption.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Study A: Microscopic changes noted in male and female livers consisted of either centrilobular or diffuse hepatocellular hypertrophy, consistent with peroxisomal proliferation. There were also dose related increases in incidence and severity of histologic changes in male rat kidneys. These changes included accumulation of dark orange or eosinophilic granular cytoplasmic pigment in cortical tubules, cortical tubular degeneration, and increased incidence of granular casts in renal tubules in high dose males. These findings were consistent with alpha 2 u-globulin nephropathy. No histopathologic changes were observed in females. The only other microscopic change observed was thymic atrophy in females from the 0.8% group, judged to have been a secondary, stress related phenomenon.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
Study A: There were no differences observed in mating, male or female fertility, gestational index, or length of gestation. Further, there were no differences in mean litter size or sex ratio.
Study B: There were no differences in the various reproductive indices, live birth indices, or male/female sex ratios.

Key result
Dose descriptor:
NOAEL
Effect level:
0.8 other: %
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Remarks on result:
other: No adverse effects observed at the highest dose tested (equivalent to ca. 600 mg/kg bw/day)
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
There were no important or treatment-related clinical findings.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
Study A: All but two of the animals selected as parents for the second generation (a control male and a 0.4% group female) survived to scheduled termination.
Study B: There were no treatment-related deaths with the test material in any F1 animals.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Study A: Body weights were significantly reduced in comparison to controls among males at the 0.8% dose level throughout the study and at the 0.4% dose level at later time points (6 to14% decrease). Among the females, body weights were generally below control values (6% reduction) in the 0.8% dose group, but were only significantly different at postpartum days 10 and 14.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Study A: Adult organ weight data were similar to those noted in the P1 generation. There were significant increases in female liver weights in all dose groups and in high dose group males. Kidney weights were significantly elevated in males from all dose groups. Left ovary weights were reduced in females in the 0.8% dose group; however, the uterus weights were not significantly different when expressed as a fraction of body weight. Weights of male reproductive organs were not different from control when expressed on an absolute basis, but testes, epididymis, and seminal vesicle weights were above control values when expressed on a relative basis.
Study B: There was evidence of liver and kidney enlargement in both males and females at the 0.2% and 0.4% levels, similar to observations in the previous two-generation Study A
Gross pathological findings:
no effects observed
Description (incidence and severity):
Study A: There were no pathologic changes in sexual organs from either males or females.
Study B: There were no gross postmortem observations judged to have been related directly to treatment with the test material in any F1 animals.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Similar to the P1 animals, histopathologic alterations were noted in kidneys of treated males and in livers of treated animals of both sexes, but there were no histologic changes in other organs.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Study A: Length of estrus cycle and number of oocytes were unaffected by treatment.

Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Study A: sperm count and sperm quality indices were unaffected by treatment.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
Study A: Mating indices, fertility indices, gestational index, mean length of gestation, and mean litter size were unaffected by treatment. The sex ratio and live birth index were significantly decreased in the 0.2% dose group, but were unaffected in the 0.4% and 0.8% dose groups, suggesting that the findings in the 0.2% dose group were due to chance rather than treatment.
Study B: There were no significant differences in fertility indices, litter size, or sex ratio. Unlike the F2 offspring in Study A, no changes were observed in percent of live births at any dose level.
Key result
Dose descriptor:
NOAEL
Effect level:
0.8 other: %
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Remarks on result:
other: No adverse effects observed at the highest dose tested (equivalent to ca. 600 mg/kg bw/day)
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs of toxicity was noted in the offspring.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Study A: Offspring survival at PND 4 was significantly reduced in the 0.8% dose group (below the HCR) but was not reduced at later time points. Survival in other dose groups was generally better than the corresponding control values.
Study B: There were no differences in survival or viability at weaning during the postnatal period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Study A: Offspring body weights were also reduced in the 0.8% dose group at PND 0 and these values were statistically significant and outside the laboratory HCR. Body weights during the postnatal period were significantly below control values in the 0.8% group, but were generally within the HCR. By PND 35, the offspring body weights were similar to control values.
Study B: there were no differences in offspring body weights during the postnatal period to PND 35.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
Study A: There was a statistically significant increase in age at vaginal patency in the 0.4 and 0.8% dose groups, but overall the difference was small (≤ 2 days) and within a range of unknown biologic relevance.
Study B: There were no differences in F1 offspring developmental landmarks, anogenital distance, nipple retention, preputial separation, or vaginal patency.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Study A: Organ weights were taken from randomly selected offspring at the end of weaning. Many of the organ weights were significantly below control values in the 0.8% group when expressed on an absolute basis; on a relative basis the brain and liver weights were above control values but there were no differences in any of the sexual organs.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Study A: No gross postmortem observations were noted in the offspring.
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
Study A: Dose related enlargement of hepatocytes with cytoplasmic eosinophilia was seen in the livers of 0.4% and 0.8% dose group offspring of both sexes. No treatment related microscopic findings were noted in kidneys or other organs.
Other effects:
no effects observed
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
0.8 other: %
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental landmarks
Remarks on result:
other: No adverse effects observed at the highest dose tested (equivalent to ca. 600 mg/kg bw/day)
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Study A: F2 offspring survival at PND 1 and 4 was significantly reduced in all treatment groups and continued to be reduced in the 0.8% dose group throughout the postnatal period. These decreases in offspring survival were generally outside the laboratory HCR.
Study B: As in the first study, F2 offspring survival was significantly reduced on PND 1 and 4 in the 0.2% and 0.4% dose groups and was below laboratory HCR. Offspring survival was generally unaffected over the remainder of the postnatal period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Study A: PND 0 body weights from male F2 offspring were reduced in the 0.8% dose group. Weight gain was significantly reduced in both male and female offspring in the 0.8% dose groups for the entire postnatal period. These decreases in PND 0 body weights and F2 offspring body weights were generally below the laboratory HCR.
Study B: all treated males and females from the 0.2% and 0.4% dose groups had mean body weights that were outside the laboratory HCR on PND 0. However, none of the values was statistically significantly different from concurrent controls, and there were no consistent dose-response relationships. There were some significant reductions in F2 offspring body weights at PND 14 and 21 in the 0.2 and 0.4% dose groups (6 to 9% difference), but these values were within the HCR and were transient. There were no significant differences in 0.4% dose group female body weights at PND 28 or in male body weights at PND 35 and 42. Overall, the effect on offspring body weights was judged to have been treatment-related due to the dose-response relationship and the similarity to decreases observed in the one-generation study in the 0.5% dose group. However, because the differences were within the HCR and transient, they were not considered to be toxicologically important.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
effects observed, treatment-related
Description (incidence and severity):
Study B: There were no differences in anogenital distance, nipple retention, or vaginal patency in the F2 offspring. Preputial separation was slightly delayed in the 0.4% dose group (1.2 days). Although this difference was statistically significant, it was deemed not adverse because the magnitude was so small.
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
Study A: Histopathologic changes in livers were noted in 0.4% and 0.8% dose groups of F2 offspring, similar to those previously seen in F1 offspring.
Other effects:
no effects observed
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
0.06 other: %
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
Remarks on result:
other: The NOAEL is equivalent to ca. 50 mg/kg bw/day.
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Table 1. Selected Organ Weights of Adult Male and Female Rats from the Two-Generation Reproductive Study A (g)

 

MALES—P1 GENERATION

Dose (%)

Liver

Kidneys

Prostate

Left Testis

Right Testis

Right Total
Epididymis

Right Cauda
Epididymis

Seminal
Vesicles

0

23.48±3.18

4.62±0.40

0.816±0.145

1.87±0.18

1.90±0.17

0.8276±0.0823

0.329±0.049

2.89±0.50

0.2

24.11±3.23

5.03±0.47**

0.821±0.186

1.88±0.14

1.90±0.16

0.8314±0.0839

0.349±0.052

3.04±0.34

0.4

25.96±4.07*

5.51±0.60**

0.796±0.143

1.87±0.20

1.87±0.20

0.8469±0.1015

0.375±0.034**

2.94±0.44

0.8

27.91±3.21**

5.71±0.52**

0.773±0.174

1.93±0.15

1.94±0.16

0.8635±0.0719

0.380±0.043**

2.85±0.44

 

MALES—P2 GENERATION

Dose (%)

Liver

Kidneys

Prostate

Left Testis

Right Testis

Right Total
Epididymis

Right Cauda
Epididymis

Seminal
Vesicles

0

23.50±3.00

4.43±0.38

0.84±0.17

2.06±0.13

2.08±0.13

0.7898±0.0708

0.223±0.046

2.85±0.41

0.2

23.81±3.53

4.82±0.55*

0.83±0.23

2.01±0.30

2.06±0.19

0.8762±0.1273

0.222±0.049

2.87±0.48

0.4

25.22±4.03

5.24±0.65**

0.86±0.19

2.00±0.21

2.02±0.22

0.8927±0.0822

0.236±0.032

3.04±0.41

0.8

26.36±3.09*

5.29±0.66**

0.80±0.14

1.99±0.17

2.00±0.18

0.9122±0.0842

0.237±0.040

2.98±0.55

 

FEMALES—P1 GENERATION

Dose (%)

Liver

Kidneys

Uterus

Right Ovary

Left Ovary

0

17.53±1.74

3.04±0.21

0.69±0.18

0.0557±0.0104

0.0636±0.0115

0.2

19.07±1.68*

3.13±0.26

0.62±0.16

0.0618±0.0131

0.0588±0.0132

0.4

20.64±1.45**

3.27±0.27*

0.66±0.20

0.0634±0.0121

0.0581±0.0102

0.8

21.00±2.02**

3.08±0.36

0.53±0.21*

0.0481±0.0118

0.0471±0.0127**

 

FEMALES—P2 GENERATION

Dose (%)

Liver

Kidneys

Uterus

Right Ovary

Left Ovary

0

21.00±1.55

3.27±0.29

0.53±0.08

0.0689±0.0122

0.0658±0.0135

0.2

24.66±2.62**

3.67±0.48**

0.64±0.13

0.0711±0.0156

0.0688±0.0113

0.4

24.82±2.99**

3.78±0.34**

0.69±0.17**

0.0699±0.0135

0.0671±0.0138

0.8

25.54±2.35**

3.54±0.35

0.49±0.15

0.0583±0.144*

0.0532±0.0165*

Values are means ± SD

*Mean significantly different from controls (P < 0.05)

**Mean significantly different from controls (P < 0.01)

Table 2. Results of Reproductive Indices, Live Birth Index, and Postnatal Survival Indices of the P1 Generation from the Two-Generation Reproductive Study A (g)

Dose (%)

Male
Mating
(%)

Male
Fertility
(%)

Female
Mating
(%)

Female
Fertility
(%)

Gestational
Index (%)

Mean
Gestation
(Days)

Mean
Litter
Size

Mean
Live
Offspring

Mean
Dead
Offspring

Live
males
(%)

Live
Females
(%)

0

88.0

76.0

88.0

81.8

89.5

22.3

15.3

15.1

0.2

48.8

51.2

0.2

93.3

76.7

93.3

78.6

100.0

22.4

14.7

14.3

0.3

54.8

45.2

0.4

80.0

76.7

80.0

91.7

100.0

22.3

14.7

14.3

0.4

48.2

51.8

0.8

88.0

86.0

88.0

90.9

100.0*

22.2

14.9

14.0

0.9

48.3

51.7

F1 OFFSPRING

Dose (%)

Live Birth
(%)

Day 1
Survival
(%)

Day 4
Survival
(%)

Day 7
Survival
(%)

Day 14
Survival
(%)

Day 21
Survival
(%)

Viability at
Weaning
(%)

0

98.7

95.5

93.9

97.8

95.5

100.0

93.4

0.2

97.6

95.8

93.0

100.0

100.0**

100.0

100.0**

0.4

96.8

94.2

91.5

99.4

99.4*

100.0

98.9*

0.8

94.2**

92.2

88.8*

98.0

98.4

100.0

96.4

HCR

95.2–99.2

96.2–100

92.8–99.7

97.8–100

93.7–100

98.8–100

86.9–100

*Mean significantly different from controls (P < 0.05)

**Mean significantly different from controls (P< 0.01)

KEY:

Male Mating Index: # males confirmed mated/#males used for mating

Male Fertility Index: # males impregnating females/# males used for mating

Female Mating Index: # females confirmed mated/# females paired

Female Fertility Index: # females pregnant (excluding unconfirmed mated females)/# females confirmed mated

Gestational Index: # females with live litters/# females pregnant

Live Birth Index: # live pups at birth/# of pups born

Day 1 Survival Index: # live pups at Day 1/# live pups at Day 0

Day 4 Survival Index: # live pups at Day 4/# live pups at Day 1

Day 7 Survival Index: # live pups at Day 7/# live pups at Day 4 (postcull)

Day 14 Survival Index: # live pups at Day 14/# live pups at Day 7

Day 21 Survival Index: # live pups at Day 21/# live pups at Day 14

Viability at Weaning Index: # live pups at Day 21/# live pups at Day 4 (post cull)

Table 3A. Time to Vaginal Patency During the Weaning Phase of the F1 Generation from the Two-Generation Reproductive Study A (days)

Dose (%)

Age at Vaginal Patency

0

32.2±1.4

0.2

32.4±2.1

0.4

33.5±2.8*

0.8

34.2±2.3**

Values are means ± SD

*Mean significantly different from controls (P < 0.05)

**Mean significantly different from controls (P < 0.01)

Table 3B. Summary of Sperm Count, Sperm Quality Indices, Estrus Cycle Length, and Oocyte Count of the P2 Generation from the Two-Generation Reproductive Study A

Dose (%)

Resistant Spermatid
Count
(106/gram of testes)

TCSC
(x 108)

Percent
Sperm
Motility

Percent
Normal
Sperm
Morphology

Estrous
Cycle Length
(Days)

Normal
Cycles
(%)

Oocyte
Counts

0

135.2

8.57

60.2

93.2

4.18

96.7

114.4

0.2

131.7

7.94

58.2

92.2

4.13

90.0

NE

0.4

129.9

8.43

60.8

95.7

4.17

90.0

NE

0.8

124.5

8.43

60.8

95.9

4.31

100.0

135.8

TCSC = Total Cauda Sperm count

NE = Not Examined

Table 4. Results of Reproductive Indices, Live Birth Index, and Postnatal Survival Indices of the P2 Generation from the Two-Generation Reproductive Study A

Dose (%)

Male
Mating
(%)

Male
Fertility
(%)

Female
Mating
(%)

Female
Fertility
(%)

Gestational
Index (%)

Mean
Gestation
(Days)

Mean
Litter
Size

Mean
Live
Offspring

Mean
Dead
Offspring

Live
males
(%)

Live
Females
(%)

0

82.8

65.5

80.0

75.0

100.0

22.4

14.3

14.1

0.2

54.3

45.7

0.2

90.0

70.0

90.0

77.8

100.0

22.4

15.2

14.4

0.8

41.7**

58.3**

0.4

83.3

66.7

83.3

76.0

100.0

22.4

14.5

14.2

0.3

51.1

48.9

0.8

100.0*

93.3*

100.0*

93.3

100.0

22.1

14.5

14.0

0.5

55.6

44.4

F2 OFFSPRING

Dose (%)

Live Birth
(%)

Day 1
Survival
(%)

Day 4
Survival
(%)

Day 7
Survival
(%)

Day 14
Survival
(%)

Day 21
Survival
(%)

Viability at
Weaning
(%)

0

98.5

96.6

94.0

99.3

99.3

100.0

98.7

0.2

94.7*

92.1*

85.8**

100.0

100.0

100.0

100.0

0.4

98.2

89.6**

86.7**

99.3

98.5*

100.0

97.8

0.8

96.8**

85.2**

77.6**

95.4*

98.4

98.9

92.9*

HCR

95.2–99.2

96.2–100

92.8–99.7

97.8–100

93.7–100

98.8–100

86.9–100

Key same as Table 2

*Mean significantly different from controls (P < 0.05)

**Mean significantly different from controls (P < 0.01)

Table 5. Selected Organ Weights of Adults and Offspring from the Two-Generation Reproductive Study B (g)

ADULTS

 

P1 Generation

P2 Generation

 

Male

Female

Male

Female

Dose (%)

Liver

Kidneys

Liver

Kidneys

Liver

Kidneys

Liver

Kidneys

0

21.27±3.04

4.14±0.36

17.57±2.54

3.07±0.31

23.61±4.06

4.64±0.6

15.93±1.60

2.98±0.26

0.02

21.03±2.59

4.06±0.38

16.95±2.37

2.97±0.19

23.22±4.22

4.68±0.47

15.85±2.02

3.01±0.26

0.06

21.31±2.57

4.12±0.35

17.41±3.48

2.98±0.26

23.42±4.01

4.64±0.58

16.12±1.61

3.07±0.30

0.2

22.26±2.68

4.36±0.43

18.21±2.68

3.07±0.30

25.15±3.48

5.09±0.54*

18.68±2.28**

3.38±0.44**

0.4

23.90±2.70**

4.74±0.45**

19.61±2.71*

3.24±0.28

26.60±3.98*

5.56±0.53*

19.54±2.47**

3.15±0.45

OFFSPRING

 

P1 Generation

P2 Generation

 

Male

Female

Male

Female

Dose (%)

Liver

Kidneys

Liver

Kidneys

Liver

Kidneys

Liver

Kidneys

0

2.38±0.51

0.72±0.10

2.18±0.35

0.70±0.08

2.00±0.47

0.65±0.11

1.90±0.35

0.65±0.11

0.02

2.35±0.41

0.74±0.09

2.14±0.27

0.71±0.07

2.15±0.42

0.71±0.13

2.10±0.49

0.70±0.13

0.06

2.26±0.47

0.70±0.13

2.00±0.47

0.68±0.14

2.11±0.41

0.67±0.10

1.89±0.38

0.63±0.10

0.2

2.39±0.40

0.72±0.10

2.24±0.42

0.73±0.12

2.08±0.44

0.66±0.11

1.95±0.42

0.62±0.10

0.4

2.32±0.58

0.72±0.13

2.17±0.47

0.68±0.11

1.93±0.54

0.63±0.12

2.05±0.54

0.63±0.11

Values are means ± SD

*Mean significantly different from controls (P < 0.05)

**Mean significantly different from controls (P < 0.01)

Table 6. Results of Reproductive Indices, Live Birth Index, and Postnatal Survival Indices of the P1 Generation from the Two-Generation Reproductive Study B

Dose (%)

Male
Mating
(%)

Male
Fertility
(%)

Female
Mating
(%)

Female
Fertility
(%)

Gestational
Index (%)

Mean
Gestation
(Days)

Mean
Litter
Size

Mean
Live
Offspring

Mean
Dead
Offspring

Live
males
(%)

Live
Females
(%)

0

90.0

86.7

90.0

92.6

10.0

22.6

14.7

14.2

0.5

50.3

49.7

0.02

90.0

96.7

90.0

100.0

96.6

22.5

14.2

14.0

0.2

46.2

53.8

0.06

79.3

89.7

79.3

100.0

92.3

22.4

15.5

15.4

0.1

50.0

50.0

0.2

96.7

86.7

96.7

89.7

100.0

22.4

14.7

14.0

0.6

49.9

50.1

0.4

83.3

80.0

83.3

84.0

100.0

22.3

14.8

14.6

0.1

52.4

47.6

F1 OFFSPRING

Dose (%)

Live Birth
(%)

Day 1
Survival
(%)

Day 4
Survival
(%)

Day 7
Survival
(%)

Day 14
Survival
(%)

Day 21
Survival
(%)

Viability at
Weaning
(%)

0

96.3

97.8

96.2

99.5

100.0

100.0

99.5

0.02

98.5

98.5

97.8

100.0

99.5

99.5

99.0

0.06

99.2*

98.4

95.9

99.5

100.0

100.0

99.5

0.2

95.8

95.9

95.3

100.0

100.0

100.0

100.0

0.4

99.2*

97.7

96.9

99.5

100.0

100.0

99.5

HCR

95.2–99.2

95.5–100

88.9–99.5

92.8–100

93.7–100

98.8–100

86.9–100

*Mean significantly different from controls (P < 0.05)

Table 7. Time to Offspring Developmental Landmarks from the Two-Generation Reproductive Study B

F1 OFFSPRING

Dose (%)

Anogential distance at PND 0 (mm)

Nipple retention at PND 12–13
(Number of thoracic nipples)

Age at Preputial
Separation (Days)

Age at Vaginal
patency (Days)

Males

Females

Males

Females

0

1.98±0.39

0.90±0.17

0±0.00

6.01±0.10

43.4±2.3

31.9±0.8

0.02

2.02±0.36

0.92±0.17

0±0.00

6.00±0.00

43.0±1.7

31.7±1.3

0.06

1.98±0.35

0.91±0.15

0±0.00

6.00±0.00

43.0±2.0

31.5±1.2

0.2

2.05±0.34

0.93±0.16

0±0.00

5.97±0.30

43.3±1.7

31.9±1.6

0.4

2.00±0.35

0.94±0.17

0±0.00

6.00±0.00

43.7±2.2

31.9±1.2

F2 OFFSPRING

Dose (%)

Anogential distance at PND 0 (mm)

Nipple retention at PND 12–13
(Number of thoracic nipples)

Age at Preputial
Separation (Days)

Age at Vaginal
patency (Days)

Males

Females

Males

Females

0

2.30±0.30

1.05±0.16

0±0.00

6.01±0.10

44.7±2.8

32.1±1.1

0.02

2.31±0.26

1.01±0.15

0±0.00

6.00±0.00

44.0±1.6

31.9±1.1

0.06

2.23±0.29

1.02±0.14

0±0.00

6.00±0.00

44.6±1.9

31.6±0.9

0.2

2.33±0.30

1.05±0.15

0±0.00

6.00±0.00

45.2±2.0

32.1±1.2

0.4

2.31±0.31

1.06±0.16

0±0.00

6.03±0.23

45.9±2.6*

32.2±1.4

Values are means ± SD

*Mean significantly different from controls (P < 0.05)

Table 8. Results of Reproductive Indices, Live Birth Index, and Postnatal Survival Indices of the P2 Generation from the Two-Generation Reproductive Study B

Dose (%)

Male
Mating
(%)

Male
Fertility
(%)

Female
Mating
(%)

Female
Fertility
(%)

Gestational
Index (%)

Mean
Gestation
(Days)

Mean
Litter
Size

Mean
Live
Offspring

Mean
Dead
Offspring

Live
males
(%)

Live
Females
(%)

0

90.0

83.3

90.0

88.9

100.0

22.3

15.9

15.6

0.4

50.4

19.6

0.02

90.0

86.7

90.0

96.3

96.2

22.3

15.2

15.0

0.2

52.3

47.7

0.06

93.3

83.3

93.3

89.3

100.0

22.4

15.6

15.2

0.4

51.8

48.2

0.2

86.7

70.0

86.7

76.9

100.0

22.3

14.9

14.8

0.1

53.9

46.1

0.4

96.7

90.0

96.7

93.1

100.0

22.1

15.9

14.9

0.7

53.0

47.0

F2 OFFSPRING

Dose (%)

Live Birth
(%)

Day 1
Survival
(%)

Day 4
Survival
(%)

Day 7
Survival
(%)

Day 14
Survival
(%)

Day 21
Survival
(%)

Viability at
Weaning
(%)

0

97.7

99.0

97.7

98.5

95.4

100.0

94.0

0.02

98.7

98.4

96.8

99.0

99.5*

100.0

98.5

0.06

97.4

97.4

96.6

99.0

100.0*

99.5

98.5*

0.2

99.4

95.2**

92.3**

98.8

98.8

98.7

96.3

0.4

95.5

89.1**

84.8**

99.0

98.5

98.5

96.0

HCR

95.2–99.2

95.5–100

88.9–99.5

92.8–100

93.7–100

98.8–100

86.9–100

Key same as Table 2

*Mean significantly different from controls (P < 0.05)

**Mean significantly different from controls (P < 0.01)

Conclusions:
In 2 two-generation reproduction toxicity studies, di-isodecyl phthalate (DIDP) was determined to have a NOAEL of 0.8% (approximately 600 mg/kg/day) for fertility, reproduction function and developmental landmarks and 0.06% (approximately 50 mg/kg/day) for F2 offspring survival.

Executive summary:

Reproductive toxicity of di-isodecyl phthalate (DIDP) was evaluated in 2 two-generation reproductive toxicity studies in accordance with EU Method B.35 and EPA OPPTS 870.3800 and GLP standards. In the first study, dietary levels of 0.2%, 0.4% and 0.8% DIDP were administered in 30 Sprague-Dawley rats per sex and per dose of generation P1 and generation P2. A second study with lower dietary levels of 0.02, 0.06, 0.2, and 0.4% DIDP was performed similarly to the first study. In both studies, there were no changes in reproductive indices and no effects on fertility in the P1 or P2 generation. There were decreases on adult body weight, but no consistent clinical signs and no mortality. In both studies, liver and kidney effects were observed in the P1 generation. Increased liver weights and associated hepatocellular hypertrophy were observed at dietary concentrations of 0.4% and greater in both studies. These dietary concentrations also produced kidney effects that were associated with alpha 2u-globulin toxicity, a male rat specific effect and thus not relevant to humans. There were no effects on live birth index, but reduced offspring survival was observed at postnatal days 1 to 4. This reduced survival was more pronounced in the F2 generation in which statistical significance was achieved at levels of 0.2% DIDP and greater. Because of these results observed in the first study, as well as the apparent lack of correlation with other treatment-related effects, a second study was carried out but the same results were obtained in F2 survival. There were also transient decreases in offspring body weights prior to weaning, corresponding to rapid offspring growth, and high levels of food consumption. There were no notable alterations in developmental landmarks. In conclusion, these studies provided a NOAEL of 0.06% (approximately 50 mg/kg/day) for F2 offspring survival and 0.8% (approximately 600 mg/kg/day) for fertility, other measures of reproductive function, and developmental landmarks.

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
EU Method B.35 (Two-Generation Reproduction Toxicity Test)
Version / remarks:
EC Dangerous Substances Directive (67/548/EEC), Annex V, Part B
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
EPA OTS 798.4700 (Reproduction and Fertility Effects)
Version / remarks:
United States Environmental Protection Agency, 40 CFR Part 798, Toxic Substances Control Act (TSCA).
Deviations:
not specified
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Crl:CD BR - VAF/PLUS®
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Stone Ridge, NY, USA.
- Weight at study initiation: 247.8-249.2 g (average in males); 178.0-180.3 g (average in females)
- Housing: rats were individually housed in suspended stainless steel and wire mesh cages.
- Diet (e.g. ad libitum): certified rodent chow (PMI Feeds, Richmond, IN, USA), ad libitum.
- Water (e.g. ad libitum): automatic watering system, ad libitum.
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 68-76ºF
- Humidity (%): 40-70 %
- Photoperiod: 12 hrs dark / 12 hrs light
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): biweekly (as DINP was confirmed stable in feed for at least 14 days)
- Mixing appropriate amounts with (Type of food): certified rodent chow (PMI Feeds, Richmond, IN, USA)
- Storage temperature of food: Not reported

Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: The mating period ended when all females were confirmed mated or approx. 3 weeks had elapsed.
- Proof of pregnancy: copulatory plug or sperm in a vaginal rinse referred to as gestational day (GD) 0.
- Further matings after two unsuccessful attempts: Not specified
- After successful mating each pregnant female was caged (how): After confirmation of mating, each mated female was returned to its own cage.
- Any other deviations from standard protocol: N/A
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The purity and stability of the test material were verified by gas chromatography and infrared analysis. Stability data confirmed that DINP was stable in feed at room temperature for at least 14 days.
Dietary analysis over the course of the study indicated concentration values of DINP to be 0.193 ± 0.008%, 0.390 ± 0.011%, and 0.782 ± 0.038% in comparison to target levels of 0.2, 0.4 and 0.8%.
Duration of treatment / exposure:
Males P1 and P2: 10 weeks prior to mating, through mating and until the day prior to euthanasia, following delivery of the last litter they sired.
Females P1 and P2: 10 weeks prior to mating, through mating, gestation and lactation and until sacrifice after weaning of offspring animals on PND 21.
Frequency of treatment:
constant exposure 7 days/week
Details on study schedule:
- Selection of parents from F1 generation when pups were 21 days of age.
Dose / conc.:
0 other: %
Remarks:
nominal in diet
Dose / conc.:
0.2 other: %
Remarks:
nominal in diet
Dose / conc.:
0.4 other: %
Remarks:
nominal in diet
Dose / conc.:
0.8 other: %
Remarks:
nominal in diet
No. of animals per sex per dose:
P1 and P2 = 30
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale:
Dietary Levels (%) were picked based on results from a preliminary one generation study where groups of 30 rats of each sex were given DINP in the diet at doses of 0, 0.5, 1, and 1.5%. Adult liver and kidney weights were elevated at 0.5% and body weight gain was reduced at 1% and 1.5%. Statistically significant effects on offspring survival, postnatal Day 0 (PND 0) weights, and weight gain during lactation were also noted. It was anticipated that body weight gain of both adults and offspring would be significantly reduced at the 0.8% level.
Positive control:
N/A
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once per week; during gestation and lactation body weights were recorded on gestation day 0, 7, 14 and 21 and postnatal days 0, 4, 7, 14 and 21.

BODY WEIGHT: Yes
- Time schedule for examinations: once per week; during gestation and lactation animals were examined on gestation day 0, 7, 14 and 21 and postnatal days 0, 4, 7, 14 and 21.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): yes. Parental food consumption was measured weekly except during gestation when female food consumption was measured at three to four day intervals.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

Oestrous cyclicity (parental animals):
Not examined
Sperm parameters (parental animals):
Parameters examined in male parental: weight of testis, epididymis, prostate and seminal vesicles.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 4/sex/litter (as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1/F2 offspring:
Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals following delivery of the last litter they sired.
- Maternal animals: All surviving animals after the last litter of each generation was weaned.

GROSS NECROPSY
- Complete gross postmortem examinations were conducted on all animals in the study.

HISTOPATHOLOGY / ORGAN WEIGHTS
Liver, kidneys (paired), testes (individual), prostate, seminal vesicles, epididymides (individual), ovaries (individual), and brain from all parental animals that survived to scheduled termination were removed and weighed. The pituitary, testes, epididymides, prostate, seminal vesicles, vagina, uterus, ovaries, mammary gland, and gross lesions from all parental animals in the control and 0.8% groups were examined microscopically.
The testes of the P1 and P2 males were preserved in Bouin’s solution. All other tissues were fixed in 10% neutral formalin. The tissues were processed, embedded in paraffin, sectioned at 5 mm, and stained with hematoxylin and eosin (H & E).
Postmortem examinations (offspring):
SACRIFICE
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
-Complete gross postmortem examinations were conducted on all animals in the study.
Statistics:
Quantitative continuous variables (body weights, food consumption, organ weights, and relative organ weights) were analyzed for statistical significance. Bartlett's test of homogeneity of variance was used to determine if the groups had equal variance at the 1% level of significance. If the variances were equivalent, the groups were compared by using a standard one-way analysis of variance (ANOVA). If significant differences among the means were indicated, Dunnet's test was performed to determine which treated groups differed from control. In addition to the ANOVA, a standard regression analysis for linear response in the dose groups was performed. If the groups did not have equivalent variances at the 1% level, then a Kruskal-Wallis test (nonparametric) was used to assess differences in group means. If the means were different, Dunn's Rank Sum test (nonparametric) was used to determine which treatment groups differed significantly from control. In addition to the Kruskal-Wallis test, Jonckheere's test (nonparametric) for ordered response was also performed.
Pup weight was analyzed by a mixed model of covariance with pups nested within dams, dams nested within dose, and total litter size as the covariant. The mathematical model incorporates the litter size as an independent variable related to pup weight. If body weight differences in groups were identified, the least squares means and the least significant difference test were used to determine which groups differed from the control group. Pup weights were analyzed separately by sex.
Parental reproductive and offspring survival incidence data were evaluated by chi-square test analysis to determine if the proportions of incidences differed between the groups tested. Each treatment group was compared to the controls by using a 2x2 Fisher Exact test. Armitage's test for linear trend in the dosage groups was also performed. All tests were reported at the 5% or 1% level of significance.
Reproductive indices:
Male mating index: no. males confirmed mated/no. males used for mating.
Male fertility index: no. males impregnating females/no. males used for mating.
Female fertility index: no. females confirmed mated/no. females paired.
Female fecundity index: no. females pregnant (excluding unconfirmed mated females)/no. females confirmed mated.
Gestational index: no. females with live litters/no. females pregnant.
Offspring viability indices:
Live birth index: no. live pups at birth/no. of pups born.
Day 1 survival index: no. live pups at Day 1/no. live pups at Day 0.
Day 4 survival index: no. live pups at Day 4/no. live pups at Day 0.
Day 7 survival index: no. live pups at Day 7/no. live pups at Day 4 (postcull).
Day 14 survival index: no. live pups at Day 14/no. live pups at Day 7.
Day 21 survival index: no. live pups at Day 21/no. live pups at Day 14.
Viability at weaning index: no. live pups at Day 21/no. live pups at Day 4 (postcull).
Clinical signs:
no effects observed
Description (incidence and severity):
There were no important or dose-dependent clinical signs of toxicity in either P1 males or females.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There was no apparent effect of treatment on survival. All but two parental (P1) animals (one male from the 0.4% group and one female from the 0.2% group) survived to scheduled termination.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights were unaffected during premating and mating and, among the females, gestation. However, dams in the 0.8% group had significantly reduced body weights at postpartum Days 14 and 21.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopic examination of the livers of parental animals revealed a minimal to moderately increased cytoplasmic eosinophilia in males and females from all treatment groups. The affected hepatocytes, although rarely enlarged microscopically, had a finely granular to homogenous cytoplasm, which in comparison to controls, was intensely eosinophilic when stained. This type of hepatocellular change, although not diagnostic, is associated with peroxisome proliferation which is considered to be a physiologic adaptation and neither adverse nor biologicly significant.
No histologic changes were seen in the testes or accessory organs or any of the other organs.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Absolute and relative weights of testes, epididymides, prostate, and seminal vesicles were unaffected.
Reproductive performance:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in male mating, male fertility, female fertility, female fecundity, or gestational indices.
There were no differences among groups in the mean length of gestation.
Sex ratios were unaffected by treatment.
However, mean litter sizes of the three treated groups were statistically significantly greater than the control group. Each treatment group contained 15 to 20% more pups than the controls between birth and culling (PND 4).
Key result
Dose descriptor:
NOAEL
Effect level:
0.8 other: %
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Remarks on result:
other: No Effects observed at the highest dose tested (ca. 500 mg/kg bw/day)
Key result
Dose descriptor:
NOAEL
Effect level:
0.2 other: %
Based on:
test mat.
Sex:
male
Basis for effect level:
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Remarks on result:
other: The calculated NOAEL was ca. 110 mg/kg bw/day.
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
Exposure to DINP did not result in any unscheduled treatment-related clinical signs in P2 males or females.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
Exposure to DINP did not result in any unscheduled deaths in P2 males or females.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weights of males in the 0.4% DINP group were 5-6% below control values but only achieved statistical significance sporadically. The mean female body weights in the same group were about 6% below control values but not significantly different from controls at any time after the first week. At the 0.8% level, the mean body weights of the males were significantly below control values throughout the pre-mating period, but the difference narrowed from ca. 13 to 7%. Among the females at the 0.8% level, the mean body weights were ca. 10% below and statistically different from control values at Week 0 and remained below control values during pre-mating, although the differences were not significantly different after the third week. This pattern continued through gestation. During lactation, the body weights of the dams were significantly below control values in the 0.8% group.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The organ weight data were similar to, but somewhat less pronounced, than those in the first generation.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No gross effects were noted during necropsy.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopic examination of the livers of parental animals revealed a minimal to moderately increased cytoplasmic eosinophilia in males and females from all treatment groups. The affected hepatocytes, although rarely enlarged microscopically, had a finely granular to homogenous cytoplasm, which in comparison to controls, was intensely eosinophilic when stained. This type of hepatocellular change, although not diagnostic, is associated with peroxisome proliferation which is considered to be a physiologic adaptation and neither adverse nor biologicly significant.
Histopathologic examination of the kidneys revealed an increased incidence of minimal to moderate renal pelvis dilatation (primarily right kidney) in P2 males in the 0.4 and 0.8% groups. This finding, which was not observed in females, was most likely related to the induction of alpha 2u-globulin.
No histologic changes were seen in the testes or accessory organs or any of the other organs.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Absolute and relative weights of testes, epididymides, prostate, and seminal vesicles were unaffected.
Reproductive performance:
no effects observed
Description (incidence and severity):
The data from the second generation were similar to those from the first. There were no effects on any of the classic reproductive indices.
The mean litter size of each of the treatment groups was significantly greater than controls.
There were no differences in fraction of live births or sex ratio.
Key result
Dose descriptor:
NOAEL
Effect level:
0.8 other: %
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Remarks on result:
other: No Effects observed at the highest dose tested (ca. 500 mg/kg bw/day)
Key result
Dose descriptor:
NOAEL
Effect level:
0.2 other: %
Based on:
test mat.
Sex:
male
Basis for effect level:
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Remarks on result:
other: The calculated NOAEL was ca. 110 mg/kg bw/day.
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
There were no important or dose-dependent clinical signs of toxicity in any of the offspring.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
Survival during lactation were unaffected by treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
PND 0 weights were significantly reduced in male offspring from the 0.8% group but were within the historical control range. Significant reductions in body weights were also noted in the 0.4% and 0.8% groups on PND 7 and 14 and in all three treatment groups at PND 21, but all of the weights were within the historical control range.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
no effects observed
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
LOEL
Generation:
F1
Effect level:
0.2 other: %
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: The calculated LOEL is 250 mg/kg bw/day.
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
There were no important or dose-dependent clinical signs of toxicity in any of the offspring.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
Survival through lactation was not different in the 0.8% dose group. Survival indices were generally comparable between groups. No differences between treated and control pups were noted during the gross necropsy at culling.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
PND 0 offspring weights in the second generation were not significantly different from controls when litter size differences were taken into account, but were below the historical control range in the 0.8% group.
Offspring body weights during lactation were significantly reduced in the 0.4% and 0.8% groups by comparison to control values. Also at PND 7 it was observed a significant reduction in the females of the 0.2% group.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No gross effects related to treatment were recorded at necropsy.
Histopathological findings:
not examined
Other effects:
no effects observed
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
LOEL
Generation:
F2
Effect level:
0.2 other: %
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: The calculated LOEL is 250 mg/kg bw/day.
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Table 5. Mean body weights of male and female rats during the premating, gestational, and postpartum phases of the first generation from the two-generation reproductive toxicity study of DINP (g)

Premating

Males

Dietary
level
(%)

Day 0

Day 7

Day 14

Day 21

Day 28

Day 35

Day 42

Day 49

Day 56

Day 63

Day 70

0

248.0±14.2

305.1±17.9

348.7±24.6

385.5±31.8

418.6±37.6

451.3±43.4

471.7±48.6

493.9±53.4

510.3±59.2

527.8±62.5

546.9±66.7

0.2

249.2±11.7

308.0±14.6

352.2±18.2

392.8±23.2

420.5±26.8

453.4±30.5

473.5±33.7

495.1±35.4

517.8±38.1

529.1±39.7

547.8±41.3

0.4

248.0±12.5

306.7±16.7

348.6±23.5

382.6±28.8

414.6±33.6

443.8±38.6

466.0±38.9

488.5±42.0

507.9±43.2

519.6±46.4

538.0±48.1

0.8

247.8±11.6

307.7±15.5

351.9±19.0

391.3±24.9

420.4±29.9

452.4±33.7

468.9±36.5

487.4±39.0

508.5±42.1

521.7±44.5

537.7±47.2

Females

0

178.0±10.1

204.7±12.6

225.5±14.1

247.7±18.6

263.0±18.6

278.1±20.0

284.8±20.4

297.6±21.4

304.7±23.1

309.6±23.9

318.2±24.1

0.2

180.3±10.0

208.7±11.7

229.9±12.6

254.1±14.2

270.5±15.3

289.2±16.3

294.7±17.3

305.2±19.0

316.0±19.5

323.3±20.8

333.5±22.3

0.4

179.0±8.8

207.7±13.4

228.4±17.7

250.2±19.1

266.2±22.0

282.6±25.8

289.7±25.6

300.7±27.5

311.4±27.5

314.9±30.5

323.6±30.7

0.8

179.0±8.7

208.0±12.9

228.8±16.4

250.4±19.4

266.2±20.3

281.0±22.1

289.2±22.0

297.7±24.3

308.2±24.8

311.9±25.0

320.5±26.7

Females

Gestation

Postpartum

Dietary
level
(%)

Day 0

Day 7

Day 14

Day 21

Day 0

Day 4

Day 7

Day 10

Day 14

Day 21

0

324.9±27.4

359.3±26.3

392.5±26.8

478.2±34.4

374.4±30.3

368.4±26.2

373.3±24.5

388.8±24.8

400.1±24.5

399.9±24.1

0.2

332.8±23.9

371.5±26.7

406.4±31.4

503.9±47.8

386.9±31.5

380.3±29.2

385.1±29.3

395.8±31.1

407.0±34.2

403.6±32.2

0.4

325.0±31.7

359.5±29.5

392.8±30.1

492.2±38.9

373.0±31.2

366.2±31.2

372.3±28.7

383.2±28.8

395.9±25.8

406.7±27.4

0.8

328.8±28.0

356.6±26.6

389.6±27.0

477.1±41.2

364.4±29.6

362.9±26.9

365.1±28.0

372.3±22.8

373.4±28.8**

368.6±32.7**

Values are means ± SD.

* Mean significantly different from the control mean (P < 0.05).

** Mean significantly different from the control mean (P < 0.01).

Table 6. Selected organ weights of male and female rats of the first and second generation from the two-generation reproductive toxicity study of DINP (g)

Males-First generation

Dietary
level
(%)

Liver

Kidneys

Left testis

Right testis

Left
epididymis

Right
epididymis

Prostate and
seminal
vesicles

0

23.27±4.33

4.80±0.66

1.86±0.13

1.85±0.15

0.80±0.07

0.83±0.08

3.89±0.58

0.2

23.52±3.79

5.18±0.54

1.88±0.17

1.89±0.17

0.82±0.09

0.81±0.08

3.97±0.52

0.4

24.68±3.76

5.47±0.48**

1.89±0.15

1.91±0.17

0.81±0.09

0.83±0.11

3.58±0.50

0.8

26.94±4.07**

5.75±0.82**

1.90±0.17

1.88±0.17

0.83±0.07

0.84±0.06

3.90±0.47

Females-First generation

Dietary
level
(%)

Liver

Kidneys

Left ovary

Right ovary

0

17.58±2.44

3.10±0.32

0.065±0.018

0.064±0.017

0.2

19.42±3.40

3.36±0.36*

0.065±0.017

0.067±0.019

0.4

21.06±3.84**

3.40±0.40*

0.069±0.019

0.068±0.018

0.8

21.46±4.10**

3.35±0.42*

0.054±0.019*

0.060±0.016

Males-Second generation

Dietary
level
(%)

Liver

Kidneys

Left testis

Right testis

Left
epididymis

Right
epididymis

Prostate and
seminal
vesicles

0

28.05±4.05

5.21±0.71

2.00±0.18

1.94±0.25

0.82±0.09

0.81±0.10

4.22±0.66

0.2

29.06±5.16

5.53±0.68

2.00±0.16

2.00±0.24

0.84±0.14

0.83±0.14

4.12±0.60

0.4

28.19±4.61

5.60±0.65

1.97±0.33

1.96±0.44

0.82±0.10

0.82±0.12

4.15±0.65

0.8

29.77±3.27

5.95±0.59**

2.05±0.16

2.02±0.34

0.87±0.09

0.87±0.14

4.06±0.61

Females-Second generation

Dietary
level
(%)

Liver

Kidneys

Left ovary

Right ovary

0

18.12±2.97

3.32±0.41

0.059±0.012

0.063±0.018

0.2

19.77±5.09

3.47±0.52

0.057±0.019

0.060±0.014

0.4

20.43±4.26

3.46±0.51

0.066±0.014

0.069±0.018

0.8

21.44±4.93*

3.43±0.44

0.056±0.018

0.057±0.019

Values are means ± SD.

* Mean significantly different from the control mean (P < 0.05).

** Mean significantly different from the control mean (P < 0.01).

Table 7. Results of reproductive indices, live birth index, and postnatal survival indices of the first generation from the two-generation reproductive toxicity study of DINP

Dietary
level
(%)

Male
mating
(%)

Male
fertility
(%)

Female
fertility
(%)

Female
fecundity
(%)

Gestational
index
(%)

Mean
gestation
(days)

Mean
litter
size

Mean
live
offspring

Mean
dead
offspring

Live
males
(%)

Live
females
(%)

0

93.3

90.0

93.3

92.9

100.0

22.3

12.5

12.2

0.3

49.5

50.5

0.2

93.1

86.2

93.1

88.9

96.0

22.2

14.9*

14.6

0.3

48.0

52.0

0.4

89.7

82.8

90.0

88.9

100.0

22.2

14.8*

14.3

0.5

49.3

50.7

0.8

93.3

83.3

93.3

85.7

100.0

22.0

15.1*

14.8

0.3

47.7

52.3

Offspring

 

 

 

 

 

 

 

 

 

 

 

Dietary
level
(%)

Live
birth
(%)

Day 1
survival
(%)

Day 4
survival
(%)

Day 7
survival
(%)

Day 14
survival
(%)

Day 21
survival
(%)

Viability at
weaning
(%)

 

 

 

 

0

97.3

98.5

95.1

96.0

100.0

99.5

95.5

 

 

 

 

0.2

98.0

98.9

97.1

99.5*

98.9

100.0

98.4

 

 

 

 

0.4

96.9

98.5

96.8

97.8

100.0

100.0

97.8

 

 

 

 

0.8

97.9

97.6

95.1

99.0

99.0

100.0

97.9

 

 

 

 

* Mean significantly different from the control mean (P < 0.05).

** Mean significantly different from the control mean (P < 0.01).

Table 8. Offspring body weights during the postnatal phase of the first generation from the two-generation reproductive toxicity study of DINP (g)

Males

Dietary level (%)

PND 0

PND 1

PND 4

PND 7

PND 14

PND 21

0

6.90±0.62

7.49±0.81

10.63±1.94

17.62±2.35

35.01±3.94

57.25±6.73

0.2

6.78±0.57

7.39±0.77

10.26±1.53

16.44±2.85

33.28±4.82

51.40±8.52*

0.4

6.48±0.63

7.03±0.80

9.54±1.72

15.28±3.19**

30.43±4.36**

47.95±7.94**

0.8

6.43±0.45*

7.05±0.62

9.74±1.25

15.67±1.74*

29.66±2.55**

46.52±5.15**

Historical range

6.35-7.02

6.68-7.46

8.53-11.43

13.64-18.74

28.81-36.73

44.89-60.77

Females

0

6.47±0.55

7.11±0.73

10.26±1.52

16.70±2.15

33.52±3.70

53.99±6.17

0.2

6.36±0.58

6.96±0.78

9.61±1.52

15.54±2.79

31.89±4.57

49.19±7.54*

0.4

6.16±0.59

6.67±0.75

9.24±1.65

14.21±3.21**

29.14±4.50**

45.63±7.31**

0.8

6.08±0.52

6.70±0.70

9.36±1.30

15.03±1.72*

28.41±3.10**

44.68±5.68**

Historical range

5.96-6.74

6.30-7.16

8.32-11.05

13.33-17.69

27.22-35.74

42.39-61.19

PND = Postnatal day.

Values are means ± SD.

* Mean significantly different from the control mean (P < 0.05).

** Mean significantly different from the control mean (P < 0.01).

Table 9. Mean body weights of male and female rats during the premating, gestational, and postpartum phases of the second generation from the two-generation reproductive toxicity study of DINP (g)

Males
Dietary
level
(%)
Day 0 Day 7 Day 14 Day 21 Day 28 Day 35  Day 42 Day 49 Day 56 Day 63 Day 70 Day 77
0 211.1±40.8 282.3±42.0 344.6±40.7 401.0±39.7 442.8±38.3 481.3±40.0 501.5±42.1 532.3±43.7 558.4±48.0 574.1±49.2 599.1±52.8 618.0±57.0
0.2 210.4±30.0 280.0±33.8 343.2±37.7 399.6±40.8 443.0±46.8 477.8±53.0 500.7±55.0 529.1±60.0 554.7±64.6 570.5±67.0 592.3±71.2 613.5±74.2
0.4 198.8±25.7 267.1±29.2 326.5±31.6 382.6±33.1 421.4±35.9 454.8±38.0 476.0±38.8 500.2±42.2* 526.3±47.3 541.5±49.9 558.9±53.9* 582.5±56.4
0.8 184.6±27.1** 251.1±28.0** 310.1±28.5** 368.1±27.8** 409.0±30.1** 445.8±32.2** 468.3±33.7* 492.5±36.5** 517.2±41.6* 532.3±43.9* 551.8±47.1* 572.7±49.4*
Females
0 167.5±33.4 201.3±27.3 226.9±23.0 250.8±26.2 266.9±25.8 280.1±26.0 289.4±25.5 303.6±25.2 312.8±29.3 318.1±29.0 327.9±30.1 334.8±29.9
0.2 165.6±20.6 198.0±19.2 226.2±21.4 252.6±23.0 271.9±25.0 285.8±24.2 292.9±24.3 307.2±27.6 316.1±28.3 321.5±29.9 327.9±30.5 337.6±33.3
0.4 157.1±15.2* 189.4±14.8 218.3±13.0 243.0±16.6 261.5±18.0 277.9±20.3 286.8±21.1 301.7±23.6 309.5±26.3 315.0±30.0 320.5±29.8 331.3±31.7
0.8 150.3±18.9** 185.8±18.9* 213.4±22.1* 237.5±23.5 254.4±25.5 271.2±26.4 278.2±26.8 288.1±27.4 299.3±28.8 303.3±28.3 310.3±29.6 318.6±32.9

Females
Gestation Postpartum
Dietary
level
(%)
Day 0 Day 7 Day 14 Day 21 Day 0 Day 4 Day 7 Day 10 Day 14 Day 21
0 330.9±28.2 370.0±30.6 408.3±37.3 497.3±43.4 382.4±40.3 381.9±36.2 387.9±35.8 393.5±30.0 407.9±27.7 394.2±24.2
0.2 333.0±31.7 365.8±37.0 402.3±34.6 497.8±40.0 375.5±38.7 376.0±34.9 377.9±34.8 388.4±34.1 398.7±37.2 389.2±32.7
0.4 326.0±25.0 360.7±25.6 397.8±29.6 492.5±38.0 374.6±35.1 364.1±38.1 368.1±37.8 380.4±27.5 394.3±28.2 386.5±29.5
0.8 315.1±37.1 342.7±36.5 374.9±38.6* 469.4±45.3 351.2±35.4 350.9±32.6* 355.0±31.4* 358.2±32.0** 364.7±33.1** 362.2±27.9**

Values are means ± SD.

* Mean significantly different from the control mean (P < 0.05).

** Mean significantly different from the control mean (P < 0.01).

Table 10. Results of reproductive indices, live birth index, and postnatal survival indices of the second generation from the two-generation reproductive toxicity study of DINP

Dietary
level
(%)
Male
mating
(%)
Male
fertility
(%)
Female
fertility
(%)
Female
fecundity
(%)
Gestational
index
(%)
Mean
gestation
(days)
Mean
litter
size
Mean
live
offspring
Mean
dead
offspring
Live
males
(%)
Live
females
(%)
0 90.0 73.3 90.0 77.8 100.0 22.5 14.1 13.9 0.2 52.3 47.7
0.2 93.3 70.0 93.3 75.0 100.0 22.3 15.5* 15.0 0.5 50.5 49.5
0.4 83.3 70.0 83.3 80.0 100.0 22.4 14.8* 14.7 0.1 50.0 50.0
0.8 80.0 63.3 80.0 70.8 100.0 22.2 15.4* 15.3 0.1 49.8 50.2
Offspring                      
Dietary
level
(%)
Live
birth
(%)
Day 1
survival
(%)
Day 4
survival
(%)
Day 7
survival
(%)
Day 14
survival
(%)
Day 21
survival
(%)
Viability at
weaning
(%)
       
0 98.7 96.7 94.8 98.8 100.0 100.0 98.8        
0.2 96.9 98.7 98.1* 99.4 100.0 99.4 98.8        
0.4 99.4 95.5 94.2 94.0* 100.0 99.4 93.4*        
0.8 99.7 97.3 96.6 98.7 100.0 100.0 98.7        

* Mean significantly different from the control mean (P < 0.05).

** Mean significantly different from the control mean (P < 0.01).

Table 11. Offspring body weights during the postnatal phase of the second generation from the two-generation reproductive toxicity study of DINP (g)

Males
Dietary level (%) PND 0 PND 1 PND 4 PND 7 PND 14 PND 21
0 6.67±0.78 7.30±1.01 10.63±1.99 18.08±3.18 37.09±4.68 62.34±7.68
0.2 6.49±0.59 7.12±0.82 10.05±1.36 16.43±2.34 34.80±3.47 57.89±6.56
0.4 6.55±0.61 7.08±0.84 9.73±1.70 15.48±2.90** 32.51±4.85** 54.82±7.45**
0.8 6.18±0.67 6.64±0.88 9.05±1.62 14.70±3.00** 29.88±4.00** 49.12±7.38**
Historical range 6.35-7.02 6.68-7.46 8.53-11.43 13.64-18.74 28.81-36.73 44.89-60.77
Females            
0 6.44±0.74 7.10±0.99 10.48±1.80 17.47±2.88 35.89±4.12 59.37±7.70
0.2 6.13±0.65 6.75±0.88 9.60±1.43 15.72±2.22* 33.64±3.66 55.50±6.36
0.4 6.11±0.61 6.59±0.87 9.05±1.71** 14.56±3.03** 31.22±4.81** 51.98±7.48**
0.8 5.92±0.72 6.41±0.92 8.68±1.70** 13.76±2.49** 28.20±3.32** 46.20±6.50**
Historical range 5.96-6.74 6.30-7.16 8.32-11.05 13.33-17.69 27.22-35.74 42.39-61.19

Values are means ± SD.

* Mean significantly different from the control mean (P < 0.05).

** Mean significantly different from the control mean (P < 0.01).

Conclusions:
In a two-generation reproductive toxicity study, di-isononyl phthalate (DINP) had no effects on fertility or testicular development in Sprague–Dawley rats. Reproductive performance was unaffected by exposure to DINP at concentrations up to 0.8% in the diet (approximately 500 mg/kg/day).
Executive summary:

The potential reproductive toxicity of di-isononyl phthalate (DINP) was evaluated in a two-generation reproductive toxicity study in accordance with EU method B.35 and EPA guideline OTS 798.4700 and GLP standards. The test substance was administered continuously in the diet at concentrations of 0.0, 0.2, 0.4 or 0.8 % DINP to groups of 30 CRL : CD(SD)BR rats per sex and per dose throughout the two generations. These dietary Levels (%) were picked based on results from a preliminary one generation study where groups of 30 rats of each sex were given DINP in the diet at doses of 0, 0.5, 1, and 1.5%. There were no changes in any of the classic reproductive parameters, i.e. mating, male or female fertility, fecundity, gestational index, or length of gestation in either study. The overall NOAELs for these effects were the highest dietary Level (%)s tested (approx. 500 mg/kg/day). There were no testicular effects in either the P1 males, exposed as juveniles and young adults or the P2 (F1) offspring exposed in utero, through lactation, and continuously to terminal sacrifice. Offspring body weights during lactation were reduced by treatment with significant differences associated with doses of approx. 250 mg/kg/day. However, the weights were within the historical control range, and the effects were largely reversible even with continued treatment. Thus, the toxicologic significance is unclear. Adult survival was unaffected at any level of dietary DINP. Liver and kidney weights were elevated at dietary Level (%)s above approx. 110 mg/kg/day, consistent with evidence from other studies of peroxisomal proliferation at these levels. This study showed that DINP treatment does not affect fertility or male reproductive development at doses of up to approximately 500 mg/kg/day in the two generation-study and 1000 mg/kg/day in the one generation-study.

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:
not specified
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (Margate, Kent, UK)
- Age at study initiation: ca. 6 weeks
- Weight at study initiation: 145 to 228 g for males and 115 to 198 g for females.
- Housing: Animals were housed in cages in groups of four of the same sex, except during mating when males and females were pair-housed, and during gestation and weaning when females were housed individually.
- Diet (e.g. ad libitum): LAD 2 SQC, Special Diet Services, Witham, UK, ad libitum.
- Water (e.g. ad libitum): ad libitum.
- Acclimation period: 13 days


Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): LAD 2 SQC, Special Diet Services, Witham, UK. The required dietary concentrations of both substances were achieved by direct dilution of a premix with untreated diet.
- Storage temperature of food: Not reported

Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Up to 3 weeks
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as Gestation Day (GD) 0.
- Further matings after unsuccessful attempts: Males that failed to mate were replaced with another of proven fertility in order to maximise the number of litters.
- After successful mating each pregnant female was caged: Individually
- Any deviations from standard protocol: No
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Tests indicated that the method of preparation produced a homogenous distribution of test substance, which was stable under the conditions used. Samples of diet were analysed for each group at weeks 1, 8 (control and 1.0% only), 11, 14, 18, 28, and 31. All formulations were within 8.5% of the nominal concentrations.
Duration of treatment / exposure:
F0 generation received the treated diet for 10 weeks before pairing and throughout mating, gestation and lactation until they were killed, males when the majority of litters had reached 2 weeks of age and females after their litters had been weaned.
F1 generation were treated from postnatal day (PND) 25 as described above for F0 generation.
Frequency of treatment:
continuous (diet)
Details on study schedule:
- F1 parental animals not mated until 10 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 25 days of age (PND 25).
- Age at mating of the mated animals in the study: ca. 14 weeks

Dose / conc.:
0 other: %
Remarks:
nominal in diet
Dose / conc.:
0.1 other: %
Remarks:
Nominal in diet
Dose / conc.:
0.5 other: %
Remarks:
nominal in diet
Dose / conc.:
1 other: %
Remarks:
nominal in diet (from week 7 of F0 generation treatment)
Dose / conc.:
2 other: %
Remarks:
nominal in diet (from week 1 to week 6 of F0 generation treatment)
No. of animals per sex per dose:
F0 generation: 28
F1 generation: 28

Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale:
A preliminary study was conducted by using dietary levels of 0.1%, 0.5%, and 1.0% of D911P. There were no apparent treatment-related effects on either the F0 or F1 generation. Therefore, dietary levels for the main study were set at 0.1%, 0.5% and 2.0% D911P. Because of marked reaction to treatment among the F0 males, the highest treatment level was reduced to 1.0% D911P after the sixth week of treatment.
Positive control:
N/A
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Not reported

BODY WEIGHT: Yes
- Time Schedule for examinations:: weekly, except for mated females, which were weighed on Days 0, 6, 13 and 20 after mating, and Days 1, 4, 7, 14 and 21 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, food consumption was monitored weekly, except for mated females for whom it was monitored individually on Days 0 through 5, 6 though 12, and 13 through 20 of gestation, and Days 1 through 6, 7 through 13, and 14 through 21 of lactation.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes


Oestrous cyclicity (parental animals):
The duration and regularity of the oestrus cycle was established by daily vaginal smears, commencing 10 days before pairing.
Sperm parameters (parental animals):
Parameters examined in all (F0 and F1) male parental generations: testis weight, epididymis weight, sperm motility, sperm count in epididymides, sperm morphology, homogenisation resistant spermatids, microscopic pathology of epididymides and testis
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F0 and F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities.

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals killed as soon as possible after the last litters in each generation were produced.
- Maternal animals: All surviving animals killed after the last litter of each generation was weaned.

GROSS NECROPSY
- Detailed gross necropsy consisted of external and internal examinations including the cranial, thoracic, abdominal and pelvic cavities and their viscera.

ORGAN WEIGHTS
The following tissues from parental animals were weighed: adrenal glands, brain, epididymides, kidneys, liver, ovaries, prostate, seminal vesicles and coagulation gland, spleen, testes, thymus, uterus with cervix.

HISTOPATHOLOGY
The following tissues/organs from parental animals were preserved for histopathology: abnormalities, adrenal glands, brain, epididymides, kidneys, liver, mammary glands, ovaries, oviduct, pituitary, prostate, seminal vesicles and coagulation gland, spleen, testis (right), thymus, uterus with cervix, vagina.
In addition, livers of five males of F1 generation were analysed for protein concentration and cyanide-insensitive palmitoyl CoA oxidase activity.
Postmortem examinations (offspring):
SACRIFICE
- F1 animals not selected for continuation of the study were killed at 25 days of age. F1 offspring were killed after weaning at 25 days of age.

GROSS NECROPSY
- Gross necropsy consisted of examination for evidence of disease or adverse reaction to treatment.

HISTOPATHOLOGY / ORGAN WEIGTHS
The following tissues from unselected F0 and F1 offspring were preserved for histopathology: abnormalities, brain, epididymides, liver, ovaries, oviduct, prostate, seminal vesicles and coagulation gland, spleen, testes, thymus, uterus with cervix,

Statistics:
For organ and body weight data, homogeneity of variance was assessed with Bartlett’s test. Where this was statistically significant, pair-wise comparison was performed with the Behrens–Fisher test, otherwise Dunnett’s test was used. Intergroup differences in macroscopic pathology and histopathology were assessed with Fisher’s exact test. Food-consumption and litter data were analysed with parametric tests (analysis of variance followed by Williams’ test) or nonparametric tests (Krúskal–Wallis followed by Shirley’s test) as appropriate. For litter data, the litter was generally taken as being the unit for analysis. Because most distributions were non-normal, nonparametric analyses were routinely used.
Reproductive indices:
Oestrus cycle, pre-coital interval, mating performance and fertility, gestation length, litter size, sex ratio, sperm analyses.
Offspring viability indices:
Post-implantation survival, liver birth index, viability index, lactation index.
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
During premating, the F0 males in the highest exposure group was severely affected by the originally administered doses of 2.0% D911P. Although this dietary concentration was reduced to 1.0% after week 6, these animals did not fully recover body weight compared to the control group. Moreover, F1 males who were never exposed to 2.0% D911P, showed reduced body weight gain after the onset of maturity. Females were less susceptible than the males. Only the highest dose group was significantly affected by treatment but was still within 10% of control values.
Body weight gains throughout the whole of gestation were not significantly affected by treatment and body weights were generally similar in all groups.
Body weights of females exposed to D911P were consistently lowered throughout lactation at the highest exposure level. Lower body weights in this group reached statistical significance within the first 2 weeks of lactation.
Body weights of F0 males at necropsy were significantly reduced by treatment at the highest dose level (19%), whereas females were largely unaffected.

Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was similar for all treatment groups during pre mating and through gestation and lactation.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The range of microscopic findings in the high-dose males was indicative of periacinar hepatotoxicity, with consequential increased cell turnover and regenerative hyperplasia. Bile duct proliferation and congestion probably resulted from the altered architecture of the liver. Again, similar effects were noted at lower incidence in males of the intermediate dosage group, and very restricted effects (periacinar vacuolation and necrosis or hypertrophy) were noted in females.
No other organs, including the reproductive organs, showed any histological changes considered to be related to treatment.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There were no apparent treatment-related effects on the duration of the oestrus cycle.
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
Testicular spermatid count was significantly higher than control in all D911P-treatment groups of the F0 generation (P < 0.05). Epididymidal weights were reduced in the highest treatment group, although epididymal sperm counts and sperm quality were unaffected.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
There were no apparent treatment-related effects on either time to mating or fecundity.
Gestation length was slightly reduced by treatment with D911P in the F0 generation.
The distribution of gestation lengths for animals exposed to 0.5% and 1.0% D911P in the F0 generation was significantly changed toward shorter duration compared to the control group. The nature of the shift in gestation length was to reduce the modal value by 0.5 days. Parturition, litter size, and offspring body weights were unaffected by treatment.
There were no effects of treatment upon the number of implantation sites, litter size, or pup survival.


Key result
Dose descriptor:
NOAEL
Effect level:
0.5 other: %
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
Remarks on result:
other: Effects observed at the highest dose level
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
After formal allocation of offspring to the F1 generation ( approx. 28 days of age), body weights in all F1 males were comparable for the first 2 or 3 weeks of treatment after weaning. However, from about the fifth week, males in the highest dose group exhibited reduced body weight gain compared to control. Females were less susceptible than the males.
Body weight gains throughout the whole of gestation were not significantly affected by treatment and body weights were generally similar in all groups.
Body weights of females exposed to D911P were consistently lowered throughout lactation at the highest and intermediate levels. Lower body weights in this group reached statistical significance within the first 2 weeks of lactation.
Body weights of F1 males at necropsy were significantly reduced by treatment at the highest dose level (12%), whereas females were largely unaffected.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was similar for all treatment groups during pre mating and through gestation and lactation.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant changes in organ weights were generally limited to animals in the highest exposure group. The exception to this was the liver in females, the weight of which was increased at dietary levels of 0.5% as well as 1.0%. Both males and females showed significant reductions in the weights of the kidneys, adrenal glands, spleen, and thymus at the highest dose level.
The weights of the testes were unaffected in all treatment groups. Epididymidal weights were reduced in the highest treatment group.
There was a significant increase in relative organ weight compared to control in testes and kidneys of males.

Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Macroscopic findings were largely limited to the livers of males in the high-dose group. Livers were small, although lobes were swollen, pale, and showed areas of macroscopic change, whereas some had irregular surfaces. Surface changes in colour and texture were noted in a number of animals. There were low incidences of similar effects in males (swelling) and females (enlargement) of the intermediate- and high-dose groups.
No other organs, including the reproductive organs, showed any macroscopic changes considered to be related to treatment.

Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The range of microscopic findings in the high-dose males was indicative of periacinar hepatotoxicity, with consequential increased cell turnover and regenerative hyperplasia. Bile duct proliferation and congestion probably resulted from the altered architecture of the liver. Again, similar effects were noted at lower incidence in males of the intermediate dosage group, and very restricted effects (periacinar vacuolation and necrosis or hypertrophy) were noted in females.
No other organs, including the reproductive organs, showed any histological changes considered to be related to treatment.
In addition, there was a significant increase in the activity of palmitoyl coenzyme A oxidase activity, a marker of peroxisomal proliferation, in the livers of F1 males in both the intermediate- and high-dose groups.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There were no apparent treatment-related effects on the duration of the oestrus cycle.

Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
Testicular spermatid counts of F1 animals exposed to D911P were not different from control. Epididymidal weights were reduced in the highest treatment group, although epididymal sperm counts and sperm quality were unaffected.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
There were no apparent treatment-related effects on either time to mating or fecundity.
Gestation length was slightly reduced by treatment with D911P in the F1 generation.
Parturition, litter size, and offspring body weights were unaffected by treatment.
There were no effects of treatment upon the number of implantation sites, litter size, or pup survival.

Key result
Dose descriptor:
NOAEL
Effect level:
0.5 other: %
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
Remarks on result:
other: Effects observed at the highest dose level
Key result
Dose descriptor:
NOAEL
Effect level:
0.1 other: %
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
There were no effects of treatment with D911P upon the number of implantation sites, litter size, or pup survival.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Pup growth was largely unaffected, except for slightly reduced body weights of offspring in the highest dosage group.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
The age of onset of sexual maturation—defined as the day at which preputial separation and vaginal opening were completed—in females was similar in all groups.
Preputial separation in males appeared to be delayed by approx. one day in the groups exposed to 1.0% D911P, but this apparent difference was not statistically significant (P > 0.05).

Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
F0 weanlings of both sexes showed increases in liver weights at PND 25, with no morphologic changes.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no macroscopic changes in offspring killed before weaning or at 25 days of age that were attributed to treatment.
Histopathological findings:
no effects observed
Other effects:
no effects observed
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
0.5 other: %
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
other: developmental landmarks
Remarks on result:
other: Effects observed at the highest dose level
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
There were no effects of treatment with D911P upon the number of implantation sites, litter size, or pup survival.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
D911P reduced body weights of F1 male and female offspring in the highest exposure group during the last 2 weeks of lactation.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
F1 weanlings of both sexes showed increases in liver weights at PND 25, with no morphologic changes.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no macroscopic changes in offspring killed before weaning or at 25 days of age that were attributed to treatment.
Histopathological findings:
no effects observed
Other effects:
no effects observed
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
0.5 other: %
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: Effects observed at the highest dose level
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Table 1. Duration of the oestrus cycle in F0 and F1 generation rats exposed to D911P in the diet

  Dietary D911P concentration (%)
F0 generation F1 generation
0 0.1 0.5 2.0/1.0 b 0 0.1 0.5 1.0
Regular c 28 26 26 26 28 28 26 28
Irregular d 1 2
Extended e
Acyclic f 1 2 2

b 2.0% from week 1 through to week 7, 1.0% thereafter

c 4–5 days duration

d one of more cycles of 2, 3 or 6–10 days duration

e at least 4 consecutive days of oestrus

f 10 days without oestrus

Table 3. Reproductive success of male and female rats exposed to D911P in the diet

  Dietary D911P concentration (%)
F0 generation F1 generation
0 0.1 0.5 2.0/1.0 a 0 0.1 0.5 1.0
Males                  
Number paired  27 28 28 28 28 28 28 28
Number mated  26b 28 28 28 28 28 27 28
Number of sires 25 26 26 26 28 27 26 25
Females                  
Number paired  28 28 28 28 28 28 28 28
Number mated  27 28 28 28 28 28 27 28
Pre-coital interval (d):                
1–4   27 26 26 25 28 28 26 26
5–8   0 1 0 2 0 0 0 1
9–12   0 1 2 0 0 0 1 1
13–16   0 0 0 1 0 0 0 0
Number pregnant  26 26 26 26 28 27 26 25
Gestation length (d) 22.8±0.5c 22.7 ± 0.4 22.5 ± 0.3§* 22.4 ± 0.4§# 22.6 ± 0.4 22.7 ± 0.4 22.4 ± 0.4 22.2 ± 0.3§†
Number littering on GD:                
21.5   1
22   4 3 6 9 4 3 11 15
22.5   8 13 14 13 15 10 8 8
23   9 8 6 2 8 12 6 1
23.5   4 2 2
24   1
Number of live litters 25d 26 26 26 28 25h 25i 24j
Implantation sites    15.3 ±3.1 15.4 ±3.2 15.5 ±2.0 14.8 ±3.2 15.7 ±2.6 13.7 ±4.0 14.5 ±3.5 13.9 ±3.9
Litter size    14.3 ±3.2 14.3 ±3.2 14.3 ±2.4 13.8 ±3.4 14.4 ±2.8 13.0 ±3.3 13.0 ±4.2 12.8 ±3.0
Live birth index (%)e  96 97 98 94 98 97 99 99
Viability index (%)f   90 89 89 86 94 93 89 98
Mean pup weight (g)                 
Male                    
PND 1    6.1 ±0.8 6.2 ±0.9 6.3 ±0.7 6.4 ±0.6 6.2 ±0.7 6.6 ±0.7 6.4 ±1.0 6.5 ±0.6
PND 7    13.4 ±3.1 13.6 ±2.9 12.9 ±3.4 13.6 ±2.5 14.1 ±2.7 14.9 ±2.2 13.5 ±3.3 13.5 ±2.1
PND 14    32.1 ±5.4 32.2 ±3.8 30.5 ±6.4 30.2 ±3.5 33.2 ±3.3 33.0 ±3.1 30.4 ±5.7 28.9 ±2.4#
PND 21    53.4 ±7.5 54.7 ±5.9 52.6 ±5.0 51.2 ±5.2 56.0 ±5.6 54.8 ±5.7 51.6 ±9.5 48.3 ±4.1#
Female                    
PND 1    5.7 ±0.7 5.8 ±0.7 5.9 ±0.6 6.0 ±0.7 5.8 ±0.6 6.1 ±0.7 6.2 ±0.9 6.2 ±0.6
PND 7    13.1 ±3.1 12.7 ±2.7 12.7 ±3.0 13.3 ±2.4 13.6 ±2.8 14.0 ±2.3 12.9 ±3.2 13.0 ±2.0
PND 14    31.4 ±5.5 30.8 ±3.6 30.5 ±3.9 29.4 ±3.6 32.3 ±3.6 31.6 ±3.4 30.2 ±3.9* 27.9 ±2.7#
PND 21    51.9 ±7.5 51.9 ±5.4 50.5 ±5.2 49.4 ±4.8 53.3 ±5.8 52.3 ±5.3 50.6 ±6.4 46.7 ±4.4#
Sex ratio (%M) PND 1 48 ±14 45 ±12 51 ±15 51 ±14 47 ±14 53 ±18 49 ±21 43 ±13
Lactation index (%)g                  
PND 7    98 95 92 98 99 95 96 100
PND 21    97 95 91 97 95 95 93 99

a 2.0% from week 1 through to week 7, 1.0% thereafter

b as a male died prior to mating one male was paired with two separate females

c mean ± SD

d one female had no live litter, one dead pup found at parturition on GD25.5

e live offspring on PND 1 compared to total litter size on PND 1

f live offspring on PND 4 compared to live offspring on PND 1

g live offspring on day of examination compared to offspring on PND 4 after culling

h two pregnant females were humanely killed on GD24 and GD24.5

i one female died at parturition

j one female had a single implant, but was not observed to give birth to a litter

* p < 0.05 compared to control

# p < 0.01 compared to control

§ distribution of gestation length significantly different to control (*, P < 0.05; #, P < 0.01; †, P < 0.001)

Table 4. Completion of preputial separation (PS) and vaginal opening (VO) in animals of the F1 generation exposed to D911P in the diet

  Dietary D911P concentration (%) Historical
Control d
Males 0 0.1 0.5 1.0
Age at PS (d) 44.8±2.3 a 44.6±2.5 44.6±2.7 46.1±2.8 43.7–47.3e
  (41–50) b (41–50) (41–50) (43–52) 41–57f
Bodyweight at PS (g) 235±22 229±27 228±23 237±26  
Females          
Age at VO (d) 34.7±2.0 35.3±2.3 34.9±3.3 35.0±2.2 33.0–35.7e
  (30–38) (32–41) (30–42) (31–41) 29–40g
Bodyweight at VO (g) 128±16 123±17 119±18 122±16  

a mean ± SD (n = 28)

b range

d laboratory historical control ranges for CD rats

e group mean values, n = 7

f individual animal values, n = 197

g individual animal values, n = 198

Table 7. Organ weights at terminal sacrifice of F0 and F1 generation male rats exposed to D911P in the diet. Liver weight data are presented in Table 10

  Dietary D911P concentration (%)
F0 generation F1 generation
0 0.1 0.5 2.0/1.0 a 0 0.1 0.5 1.0
Number of animals 27 28 28 28 28 28 28 27
Bodyweight (g)     639±71b 653±70 625±71 517±69# 611±72 621±86 634±82 538±64#
Organ weights, absolute (g)                  
adrenal gland (X10)    0.54±0.13 0.48±0.15 0.47±0.12 0.45±0.11* 0.61±0.12 0.56±0.12 0.56±0.07* 0.50±0.08#
brain      2.15±0.09 2.16±0.11 2.15±0.09 2.09±0.09* 2.18±0.09 2.18±0.15 2.18±0.10 2.15±0.09
epididymides      1.34±0.12 1.32±0.09 1.29±0.13 1.26±0.10* 1.32±0.10 1.26±0.16c 1.30±0.09 1.23±0.17*
kidneys      4.33±0.51 4.42±0.44 4.38±0.53 3.85±0.52# 4.13±0.68c 4.04±0.53 4.31±0.68 3.76±0.38*
prostate (X10)     6.53±1.69 6.49±1.83 6.52±1.77 5.55±1.05* 6.00±1.98 5.52±1.39 5.79±1.61 5.20±1.21
seminal vesicles     2.49±0.29 2.66±0.26* 2.56±0.46 2.19±0.33# 2.35±0.30 2.36±0.35c 2.28±0.27 2.05±0.28#
spleen (X10)     8.64±1.32 8.85±2.00 8.21±1.15 7.59±1.06# 8.64±1.68 8.53±1.14 8.55±1.98 7.43±1.27#
testes      3.84±0.42 3.82±0.36 3.75±0.40 3.75±0.30 3.78±0.32 3.85±0.59c 3.75±0.31 3.67±0.63
thymus (X10)     3.28±0.65 3.27±1.02 3.40±1.30 3.18±1.07 3.64±0.96 3.71±0.88 3.75±1.01 3.22±0.96
Organ weights, relative to body (%)                
epididymides      0.21±0.02 0.20±0.03 0.21±0.03 0.25±0.03# 0.22±0.03 0.21±0.03c 0.21±0.03 0.23±0.04
kidneys      0.68±0.05 0.68±0.07 0.70±0.06 0.75±0.06# 0.67±0.07c 0.65±0.06 0.68±0.07 0.70±0.07
prostate      0.10±0.03 0.10±0.03 0.10±0.02 0.11±0.02 0.09±0.03 0.09±0.02 0.09±0.03 0.10±0.03
seminal vesicles     0.39±0.05 0.41±0.06 0.42±0.10 0.43±0.07* 0.39±0.07 0.39±0.06c 0.37±0.05 0.39±0.07
testes      0.60±0.07 0.59±0.09 0.61±0.08 0.73±0.09# 0.63±0.08 0.62±0.12c 0.60±0.08 0.70±0.16*

a 2.0% from week 1 though to week 7, 1.0% thereafter

b mean ± SD

c n = 27

* P < 0.05 compared to control

# P < 0.01 compared to control

Table 8. Organ weights at terminal sacrifice of F0 and F1 generation female rats exposed to D911P in the diet. Liver weight data are presented in Table 12

  Dietary D911P concentration (%)
F0 generation F1 generation
  0 0.1 0.5 2.0/1.0 a 0 0.1 0.5 1.0
Number of animals 24 23 22 23 26 23 23 24
Bodyweight (g)     351±28b 356±27 342±20 337±24 341±31 343±34 333±23 325±24*
Organ weights, absolute (g)                  
adrenal gland (X10)    0.88±0.17 0.88±0.14 0.79±0.13 0.69±0.13# 0.87±0.11 0.82±0.11 0.77±0.11# 0.64±0.12#
brain      1.95±0.20 1.95±0.09 1.95±0.09 1.94±0.14 2.00±0.06 1.96±0.08 1.97±0.07 2.00±0.10
kidneys      3.07±0.38 3.02±0.25 3.04±0.30 2.94±0.21 2.91±0.36 2.77±0.27 2.78±0.21 2.86±0.28
Ovaries (X10)     1.10±0.22 1.21±0.20 1.11±0.19 0.98±0.21 1.10±0.15 1.17±0.16 1.11±0.16 0.98±0.20c
spleen (X10)     6.26±0.99 6.21±1.06 5.83±0.74 5.37±0.88# 6.63±0.92 6.36±0.96 5.88±0.80# 5.35±0.72#
thymus (X10)     2.28±0.59 1.97±0.61 1.84±0.65 1.75±0.66* 2.50±0.72 2.32±0.49 2.32±0.86 1.75±1.14*
uterus + cervix 0.53±0.11 0.53±0.11 0.53±0.18 0.41±0.10# 0.52±0.13 0.46±0.13 0.50±0.13 0.43±0.15
Organ weights, relative to body (%)                
kidneys      0.88±0.09 0.85±0.06 0.89±0.09 0.88±0.07 0.86±0.06 0.81±0.07 0.83±0.05 0.88±0.07*
Ovaries (X100)     3.15±0.67 3.41±0.52 3.25±0.55 2.91±0.60 3.19±0.41 3.45±0.49 3.34±0.41 3.02±0.50c
uterus + cervix 0.15±0.03 0.15±0.04 0.16±0.06 0.12±0.03# 0.15±0.04 0.13±0.04 0.15±0.04 0.13±0.05

a 2.0% from week 1 though to week 7, 1.0% thereafter

b mean ± SD

c n = 23

* P < 0.05 compared to control

# P < 0.01 compared to control

Table 10. Liver pathology data for male rats of the F0 and F1 generations and male offspring exposed to D911P

  Dietary D911P concentration (%)
F0 generation F1 generation
  0 0.1 0.5 2.0/1.0 a 0 0.1 0.5 1.0
Number of animals  27 28 28 28 28 28 28 27
Bodyweight (g)   639±71b 653±70 625±71 517±69# 611±72 621±86 634±82 538±64#
Liver weight (g)  24.2±4.5 24.7±3.5 24.3±4.0c 20.7±6.0* 23.5±4.2 24.1±4.4 25.9±6.9 19.9±3.4#f
Palmitoyl CoA oxidase (nmol/min/mg protein) d   3.9 ± 0.3 3.7 ± 0.2 5.1 ± 0.4# 7.7 ± 1.1#
Number of livers examined microscopically    28 7 11 28 28 3 12 28
Periacinar hepatocytic fatty vacuolation    9 23§ 2 8 18§
Focus of hepatocytic fatty vacuolation    12§ 6*
Congestion    1 1 1 3 3
Periacinar hepatocytic hypertrophy  1 5 20§ 5 24§
Regenerative hyperplasia   7* 18§
Periacinar hepatocytic necrosis  3 28§ 3 25§
Bile duct proliferation  1 24§ 1 1 23§
Periacinar hepatocyte pigment  1 10§
Clear cell foci  1 24§ 13§
Basophilic foci   1 22§ 16§
Eosinophilic foci   8# 3
Offspring sacrificed at PND25                
number of litters e  24 22 22 21 26 23 22 24
bodyweight (g) 70.2±10.4 70.6±7.7 69.8±7.7 67.8±8.1 73.3±7.2 71.8±6.6 70.1±8.9 64.4±5.2#
liver weight (g) 4.27±0.80 4.23±0.53 4.64±0.74 4.95±0.82# 4.36±0.55 4.47±0.48 4.88±0.83* 4.69±0.54*

a 2.0% from week 1 through to week 7, 1.0% thereafter

b mean ± SD

c n = 27

d data are for five animals

e one to four pups per litter

f n = 26

* P < 0.05 compared to control

# P < 0.01 compared to control

§ P < 0.001 Fischer’s exact test

Table 12. Liver pathology data for female rats of the F0 and F1 generations and female offspring exposed to D911P

  Dietary D911P concentration (%)
F0 generation F1 generation
  0 0.1 0.5 2.0/1.0 a 0 0.1 0.5 1.0
Number of animals  24 23 22 23 26 23 23 24
Bodyweight (g)   351 ± 28b 356 ± 27 342 ± 20 337 ± 24 341 ± 31 343 ± 34 333 ± 23 325 ± 24*
Liver weight (g)  19.9 ± 2.5 20.9 ± 2.6 24.3 ± 1.8# 25.1 ± 2.1# 20.5 ± 2.7 20.9 ± 2.7 23.4 ± 2.6# 25.2 ± 2.6#
Number of livers examined microscopically    28 2 5 28 28 2 11 28
Periacinar hepatocytic fatty vacuolation    1 8# 3 4
Periacinar hepatocytic hypertrophy  7* 2 2
Offspring sacrificed at PND25                
number of litters c 24 23 22 23 26 22 22 24
bodyweight (g) 66.4 ± 11.0 66.3 ± 7.4 65.8 ± 7.7 64.1 ± 6.3 68.7 ± 7.5 66.9 ± 6.2 66.0 ± 8.2 60.6 ± 5.4#
liver weight (g) 3.95 ± 0.75 3.90 ± 0.59 4.32 ± 0.69 4.51 ± 0.69# 4.19 ± 0.62 4.23 ± 0.43 4.71 ± 0.73* 4.51 ± 0.52*

a 2.0% from week 1 through to week 7, 1.0% thereafter

b mean ± SD

c one to five pups per litter

* P < 0.05 compared to control

# P < 0.01 compared to control

Table 13. Testicular and epididymal sperm counts and sperm quality in F0 and F1 rats exposed to D911P in the diet

  Dietary D911P concentration (%)
F0 generation F1 generation
0 0.1 0.5 2.0/1.0 a 0 0.1 0.5 1.0
Number of animals 27 28 28 28 28 27 28 27
Testis spermatid count (106/g) 108±33 b 139±49* 134±41* 136±51* 122±43 123±45 114±37 115±50
Epididymis sperm evaluation
sperm count (107/ml)
219±47 227±71 223±58 206±61 215±60 200±55 199±36 201±50
motile (%) 76±8 75±10 73 ± 11 73 ±15 77 ±8 77±8 77±6 75±8
progressively motile (%) 40±9 40± 10 38 ±9 39±10 38 ±7 38±8 40±7 37± 7
normal morphology (%) 96±3 95±4 95±3 93±11 91 ±9 92±7 89±11 89 ± 12

a 2.0% from week 1 through to week 7, 1.0% thereafter

b mean ± SD

Table 15. Pathology of the reproductive tract in male and female rats of the F0 and F1 generations exposed to D911P in the diet

  Dietary D911P concentration (%)
F0 generation F1 generation
0 0.1 0.5 2.0/1.0 a 0 0.1 0.5 1.0
Males                
Epididymides                    
small    0/28b 0/28 1/28 2/28 0/28 2/28 1/28 1/28
reduced spermc    0/28 0/2 0/2 1/28 0/28 1/1 1/1 1/28
Prostate                    
small    1/28 0/28 0/28 0/28
large     1/28 0/28 0/28 0/28
chronic inflammation    6/28 0/2 0/2 3/28 12/28 4/28*
atrophy     3/28 0/2 0/2 2/28 2/28 2/28
Seminal vesicles                   
small     1/28 0/28 0/28 1/28
misshapen    1/28 0/28 0/28 0/28
Testes                    
small    0/28 0/28 1/28 2/28 0/28 1/28 0/28 1/28
dark     0/28 0/28 1/28 2/28 0/28 0/28 1/28 0/28
flaccid     0/28 0/28 1/28 2/28 0/28 1/28 1/28 1/28
not evident            0/28 1/28 0/28 0/28
tubular dilatation    0/28 1/1 0/1 0/28 1/28 0/28
occasional germ cell depletion  1/28 0/1 0/1 0/28 2/28 1/28
generalised germ cell depletion  0/28 0/1 1/1 1/28 0/28 1/28
Females                    
Mammary glands                   
pale     0/28 3/28 1/28 2/28 2/28 2/28 2/28 0/28
secretory activity    2/3 1/3 2/3
inactive     1/2 3/3 1/1 2/3 0/3 2/3 1/3
Ovaries                    
reduced number of developing follicles 0/28 0/5 0/5 1/28
fatty vacuolation of corpora lutea 0/28 0/5 1/5 1/28
Uterus                    
fluid distension    1/28 0/28 0/28 0/28 1/28 0/28 2/28 0/28
dilated glands    6/28 1/5 0/5 0/28*        
dilated     5/28 2/5 1/5 0/28 8/28 2/5 3/5 3/28

Macroscopic findings are in italics. All organs from the control and high dose groups were examined microscopically, as well as those from the low and

intermediate treatment groups that were found abnormal under macroscopic evaluation.

a 2.0% from week 1 through to week 7, 1.0% thereafter

b number of affected/number examined

c histopathology for right epididymis/testis

— not reported for any group

* P < 0.05

Conclusions:
In a two-generation reproduction toxicity study, di-(C9-C11 alkyl) phthalate (D911P) was determined without adverse effects upon fertility or reproductive performance in rats when administered in the diet at levels that induce systemic toxicity. The NOAEL for effects upon reproductive health parameters and general systemic toxicity was 0.5% in the diet.
Executive summary:

Reproductive toxicity of di-(C9-C11 alkyl) phthalate (D911P) was evaluated in a two-generation reproductive toxicity study in accordance with EPA guideline OPPTS 870.3800 and GLP standards. The test substance was administered continuously in the diet at concentrations of 0.1, 0.5 or 1 % to groups of 28 Sprague-Dawley rats per sex and per dose for both generation F0 and generation F1. The highest dosage group animals were given 2% D911P for approximately six weeks (43 days) at the start of the F0 generation but this was reduced to 1% D911P because of a marked reduction in bodyweight gain of the males. The major treatment-related effect was a severe reduction in adult male body weight gain at the highest dose, with a lesser effect in females. There was no treatment-related deterioration of fertility or fecundity at any dose level. The only effects upon reproductive parameters attributable to treatment were a reduction in epididymides weight by 1.0% D911P. However, in the absence of functional or histopathologic changes, the significance of these observations is unclear. Ovarian function, assessed by the oestrus cycle and mating behaviour, and epididymidal sperm concentration, motility, and morphology were unaffected by the treatment. Transiently reduced body weight of offspring in both generations may be a secondary response to depression of maternal body weight gain or a direct effect of D911P in the diet resulting from pups eating it. Reductions in organ weights (except liver) are also attributed to a nonspecific response to reduced body weight. The livers of male rats, and to a lesser extent females, showed the classic biochemical, morphologic, and histologic changes that would be expected of a substance of the peroxisome proliferator class—specifically hypertrophy, hyperplasia, and an increase in the activity of palmitoyl CoA oxidase. The NOAEL for effects upon reproductive health parameters and general systemic toxicity was 0.5% in the diet. This NOAEL was based on transient depression of offspring body weight gain (males and females), reduced parental body weight gain (males), and reduced reproductive organ weights (males and females). Finally, a NOAEL for effects upon the liver was established in 0.1% D911P.

Endpoint:
two-generation reproductive toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
The target substance (DUP) belongs to the OECD HPV category High Molecular Weight Phthalate Ester (HMWPE) which consists of esters with an alkyl carbon backbone with 7 carbon (C) atoms or greater. The source substance (DIDP) although is not formally part of this category as it was assessed previously, it satisfies the category definition as its backbone length is C7 or above and also produces similar effects of developmental or reproductive toxicity. Thus, these substances share the same functional groups and also have comparable environmental and toxicological properties.
See attached the reporting format.
Reason / purpose for cross-reference:
read-across source
Key result
Dose descriptor:
NOAEL
Effect level:
0.85 other: %
Based on:
other: Read across from an analogue
Sex:
male/female
Basis for effect level:
reproductive performance
Remarks on result:
other: read-across from an analogue for which no adverse effects were observed at the highest dose tested (0.8% equivalent to ca. 600 mg/kg bw/day)
Key result
Critical effects observed:
no
Key result
Dose descriptor:
NOAEL
Effect level:
0.85 other: %
Based on:
other: Read across from an analogue
Sex:
male/female
Basis for effect level:
reproductive performance
Remarks on result:
other: read-across from an analogue for which no adverse effects were observed at the highest dose tested (0.8 % equivalent to ca. 600 mg/kg bw/day)
Key result
Critical effects observed:
no
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
0.85 other: %
Based on:
other: Read across from an analogue
Sex:
male/female
Basis for effect level:
other: developmental landmarks
Remarks on result:
other: read-across from an analogue for which no adverse effects were observed at the highest dose tested (0.8 % equivalent to ca. 600 mg/kg bw/day)
Key result
Critical effects observed:
no
Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
0.064 other: %
Based on:
other: Read across from an analogue
Sex:
male/female
Basis for effect level:
viability
Remarks on result:
other: Read-across from an analogue for which the NOAEL is 0.06%, equivalent to ca. 50 mg/kg bw/day.
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
Based on the read-across approach from the analogue diisodecyl phthalate (DIDP) assessed in 2 two-generation reproduction toxicity studies, diundecyl phthalate (DUP) is determined to have a NOAEL of 0.85% (approximately 637.69 mg/kg/day) for fertility, reproduction function and developmental landmarks and 0.064% (approximately 53.14 mg/kg/day) for F2 offspring survival.

Executive summary:

Reproductive toxicity of di-isodecyl phthalate (DIDP) was evaluated in 2 two-generation reproductive toxicity studies in accordance with EU Method B.35 and EPA OPPTS 870.3800 and GLP standards. In the first study, dietary levels of 0.2%, 0.4% and 0.8% DIDP were administered in 30 Sprague-Dawley rats per sex and per dose of generation P1 and generation P2. A second study with lower dietary levels of 0.02, 0.06, 0.2, and 0.4% DIDP was performed similarly to the first study. In both studies, there were no changes in reproductive indices and no effects on fertility in the P1 or P2 generation. There were decreases on adult body weight, but no consistent clinical signs and no mortality. In both studies, liver and kidney effects were observed in the P1 generation. Increased liver weights and associated hepatocellular hypertrophy were observed at dietary concentrations of 0.4% and greater in both studies. These dietary concentrations also produced kidney effects that were associated with alpha 2u-globulin toxicity, a male rat specific effect and thus not relevant to humans. There were no effects on live birth index, but reduced offspring survival was observed at postnatal days 1 to 4. This reduced survival was more pronounced in the F2 generation in which statistical significance was achieved at levels of 0.2% DIDP and greater. Because of these results observed in the first study, as well as the apparent lack of correlation with other treatment-related effects, a second study was carried out but the same results were obtained in F2 survival. There were also transient decreases in offspring body weights prior to weaning, corresponding to rapid offspring growth, and high levels of food consumption. There were no notable alterations in developmental landmarks. In conclusion, these studies provided a NOAEL of 0.06% (approximately 50 mg/kg/day) for F2 offspring survival and 0.8% (approximately 600 mg/kg/day) for fertility, other measures of reproductive function, and developmental landmarks. Based on these results, the read-across approach was applied and DUP is determined to have a NOAEL of 0.85% (approximately 637.69 mg/kg/day) for fertility, reproduction function and developmental landmarks and 0.064% (approximately 53.14 mg/kg/day) for F2 offspring survival.

Endpoint:
two-generation reproductive toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
The target substance (DUP) belongs to the OECD HPV category High Molecular Weight Phthalate Ester (HMWPE) which consists of esters with an alkyl carbon backbone with 7 carbon (C) atoms or greater. The source substance (DINP) although is not formally part of this category as it was assessed previously, it satisfies the category definition as its backbone length is C7 or above and also produces similar effects of developmental or reproductive toxicity. Thus, these substances share the same functional groups and also have comparable environmental and toxicological properties.
See attached the reporting format.
Reason / purpose for cross-reference:
read-across source
Key result
Dose descriptor:
NOAEL
Effect level:
0.91 other: %
Based on:
other: Read across from an analogue
Sex:
male/female
Basis for effect level:
reproductive performance
Remarks on result:
other: read-across from an analogue for which no effects were observed at the highest dose tested (0.8% equivalent to ca. 500 mg/kg bw/day)
Key result
Dose descriptor:
NOAEL
Effect level:
0.23 other: %
Based on:
other: Read across from an analogue
Sex:
male
Basis for effect level:
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Remarks on result:
other: read-across from an analogue for which the NOAEL is 0.2%, equivalent to ca. 110 mg/kg bw/day.
Key result
Critical effects observed:
no
Key result
Dose descriptor:
NOAEL
Effect level:
0.91 other: %
Based on:
other: Read across from an analogue
Sex:
male/female
Basis for effect level:
reproductive performance
Remarks on result:
other: read-across from an analogue for which no Effects were observed at the highest dose tested (0.8%, equivalent to ca. 500 mg/kg bw/day)
Key result
Dose descriptor:
NOAEL
Effect level:
0.23 other: %
Based on:
other: Read across from an analogue
Sex:
male
Basis for effect level:
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Remarks on result:
other: read-across from an analogue for which the NOAEL is 0.2%, equivalent to ca. 110 mg/kg bw/day.
Key result
Critical effects observed:
no
Key result
Dose descriptor:
LOEL
Generation:
F1
Effect level:
0.23 other: %
Based on:
other: Read across from an analogue
Sex:
female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: read-across from an analogue for which the LOEL is 250 mg/kg bw/day (0.2%).
Key result
Critical effects observed:
no
Key result
Dose descriptor:
LOEL
Generation:
F2
Effect level:
0.23 other: %
Based on:
other: Read across from an analogue
Sex:
female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: read-across from an analogue for which the LOEL is 250 mg/kg bw/day (0.2%).
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
Based on the read-across approach from the analogue Diisononyl phthalate (DINP) assessed in a two-generation reproductive toxicity study, diundecyl phthalate (DUP) is not expected to have effects on fertility or testicular development in Sprague–Dawley rats and the NOAEL for reproductive performance is estimated to be 0.91% in the diet (approximately 567.02 mg/kg/day).
Executive summary:

The potential reproductive toxicity of di-isononyl phthalate (DINP) was evaluated in a two-generation reproductive toxicity study in accordance with EU method B.35 and EPA guideline OTS 798.4700 and GLP standards. The test substance was administered continuously in the diet at concentrations of 0.0, 0.2, 0.4 or 0.8 % DINP to groups of 30 CRL : CD(SD)BR rats per sex and per dose throughout the two generations. These dietary Levels (%) were picked based on results from a preliminary one generation study where groups of 30 rats of each sex were given DINP in the diet at doses of 0, 0.5, 1, and 1.5%. There were no changes in any of the classic reproductive parameters, i.e. mating, male or female fertility, fecundity, gestational index, or length of gestation in either study. The overall NOAELs for these effects were the highest dietary Level (%)s tested (approx. 500 mg/kg/day). There were no testicular effects in either the P1 males, exposed as juveniles and young adults or the P2 (F1) offspring exposed in utero, through lactation, and continuously to terminal sacrifice. Offspring body weights during lactation were reduced by treatment with significant differences associated with doses of approx. 250 mg/kg/day. However, the weights were within the historical control range, and the effects were largely reversible even with continued treatment. Thus, the toxicologic significance is unclear. Adult survival was unaffected at any level of dietary DINP. Liver and kidney weights were elevated at dietary Level (%)s above approx. 110 mg/kg/day, consistent with evidence from other studies of peroxisomal proliferation at these levels. This study showed that DINP treatment does not affect fertility or male reproductive development at doses of up to approximately 500 mg/kg/day in the two generation-study and 1000 mg/kg/day in the one generation-study.

Based on these results, the read-across approach was applied and diundecyl phthalate (DUP) is estimated to have a NOAEL for reproductive performance of 0.91% in the diet (approximately 567.02 mg/kg/day).

Endpoint:
two-generation reproductive toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Both the target substance (DUP) and the source substance (D911P) belong to the OECD HPV category High Molecular Weight Phthalate Ester (HMWPE) which consists of esters with an alkyl carbon backbone with 7 carbon (C) atoms or greater. Thus, these substances share the same functional groups and also have comparable environmental and toxicological properties.
See attached the reporting format.
Reason / purpose for cross-reference:
read-across source
Key result
Dose descriptor:
NOAEL
Effect level:
0.53 other: %
Based on:
other: Read across from an analogue
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
Remarks on result:
other: Read-across from an analogue for which effects were observed at the highest dose level.
Key result
Critical effects observed:
no
Key result
Dose descriptor:
NOAEL
Effect level:
0.53 other: %
Based on:
other: Read across from an analogue
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
Remarks on result:
other: Read-across from an analogue for which effects were observed at the highest dose level
Key result
Dose descriptor:
NOAEL
Effect level:
0.11 other: %
Based on:
other: Read across from an analgue
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: Read-across from an analogue for which the NOAEL was 0.1% in diet
Key result
Critical effects observed:
no
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
0.53 other: %
Based on:
other: Read across from an analogue
Sex:
male/female
Basis for effect level:
body weight and weight gain
other: developmental landmarks
Remarks on result:
other: Read-across from an analogue for which effects were observed at the highest dose level.
Key result
Critical effects observed:
no
Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
0.53 other: %
Based on:
other: Read across from an analogue
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: Read-across from an analogue for which effects were observed at the highest dose level.
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
Based on the read-across approach from the analogue di-(C9-C11 alkyl) phthalate (D911P) assessed in a two-generation reproduction toxicity study, diundecyl phthalate (DUP) is considered without adverse effects upon fertility or reproductive performance in rats at dietary levels that induce systemic toxicity and the NOAEL is determined to be 0.53% in the diet.
Executive summary:

Reproductive toxicity of di-(C9-C11 alkyl) phthalate (D911P) was evaluated in a two-generation reproductive toxicity study in accordance with EPA guideline OPPTS 870.3800 and GLP standards. The test substance was administered continuously in the diet at concentrations of 0.1, 0.5 or 1 % to groups of 28 Sprague-Dawley rats per sex and per dose for both generation F0 and generation F1. The highest dosage group animals were given 2% D911P for approximately six weeks (43 days) at the start of the F0 generation but this was reduced to 1% D911P because of a marked reduction in bodyweight gain of the males. The major treatment-related effect was a severe reduction in adult male body weight gain at the highest dose, with a lesser effect in females. There was no treatment-related deterioration of fertility or fecundity at any dose level. The only effects upon reproductive parameters attributable to treatment were a reduction in epididymides weight by 1.0% D911P. However, in the absence of functional or histopathologic changes, the significance of these observations is unclear. Ovarian function, assessed by the oestrus cycle and mating behaviour, and epididymidal sperm concentration, motility, and morphology were unaffected by the treatment. Transiently reduced body weight of offspring in both generations may be a secondary response to depression of maternal body weight gain or a direct effect of D911P in the diet resulting from pups eating it. Reductions in organ weights (except liver) are also attributed to a nonspecific response to reduced body weight. The livers of male rats, and to a lesser extent females, showed the classic biochemical, morphologic, and histologic changes that would be expected of a substance of the peroxisome proliferator class—specifically hypertrophy, hyperplasia, and an increase in the activity of palmitoyl CoA oxidase. The NOAEL for effects upon reproductive health parameters and general systemic toxicity was 0.5% in the diet. This NOAEL was based on transient depression of offspring body weight gain (males and females), reduced parental body weight gain (males), and reduced reproductive organ weights (males and females). Finally, a NOAEL for effects upon the liver was established in 0.1% D911P. Based on these results, the read-across approach was applied and diundecyl phthalate (DUP) is considered without adverse effects upon fertility or reproductive performance in rats at dietary levels that induce systemic toxicity and the NOAEL is determined to be 0.53% in the diet.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
637.69 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
A weight of evidence approach has been applied. Several experimental studies are available with a Klimisch score of 2.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Weight of evidence from experimental results with analogue substances. Read across approach.

Diisodecyl phthalate (DIDP): This substance was investigated in 2 two-generation reproductive toxicity studies in accordance with EU Method B.35 and EPA OPPTS 870.3800 and GLP standards. In the first study, dietary levels of 0.2%, 0.4% and 0.8% DIDP were administered in 30 Sprague-Dawley rats per sex and per dose of generation P1 and generation P2. There were no effects on live birth index, but reduced offspring survival was observed at postnatal days 1 to 4. This reduced survival was more pronounced in the F2 generation in which statistical significance was achieved at levels of 0.2% DIDP and greater. Because of these results observed in the first study, as well as the apparent lack of correlation with other treatment-related effects, a second study was carried out with lower dietary levels of 0.02, 0.06, 0.2, and 0.4% DIDP, but the same results were obtained in F2 survival. These studies provided a NOAEL of 0.06% (approximately 50 mg/kg/day) for F2 offspring survival and 0.8% (approximately 600 mg/kg/day) for fertility, other measures of reproductive function, and developmental landmarks. Based on these results, the read-across approach was applied and DUP is determined to have a NOAEL of 0.85% (approximately 637.69 mg/kg/day) for fertility, reproduction function and developmental landmarks and 0.064% (approximately 53.14 mg/kg/day) for F2 offspring survival.

Diisononyl phthalate (DINP): This substance was evaluated in a two-generation reproductive toxicity study in accordance with EU method B.35 and EPA guideline OTS 798.4700 and GLP standards. The test substance was administered continuously in the diet at concentrations of 0.0, 0.2, 0.4 or 0.8 % DINP to groups of 30 CRL : CD(SD)BR rats per sex and per dose throughout the two generations. This study showed that DINP treatment does not affect fertility or male reproductive development at doses of up to approximately 500 mg/kg/day in the two generation-study. Based on these results, the read-across approach was applied and diundecyl phthalate (DUP) is estimated to have a NOAEL for reproductive performance of 0.91% in the diet (approximately 567.02 mg/kg/day).

Di-(C9-C11 alkyl) phthalate (D911P): This substance was evaluated in a two-generation reproductive toxicity study in accordance with EPA guideline OPPTS 870.3800 and GLP standards. The test substance was administered continuously in the diet at concentrations of 0.1, 0.5 or 1 % to groups of 28 Sprague-Dawley rats per sex and per dose for both generation F0 and generation F1. The highest dosage group animals were given 2% D911P for approximately six weeks (43 days) at the start of the F0 generation but this was reduced to 1% D911P because of a marked reduction in bodyweight gain of the males. The NOAEL for effects upon reproductive health parameters and general systemic toxicity was 0.5% in the diet. This NOAEL was based on transient depression of offspring body weight gain (males and females), reduced parental body weight gain (males), and reduced reproductive organ weights (males and females). Finally, a NOAEL for effects upon the liver was established in 0.1% D911P. Based on these results, the read-across approach was applied and diundecyl phthalate (DUP) is considered without adverse effects upon fertility or reproductive performance in rats at dietary levels that induce systemic toxicity and the NOAEL is determined to be 0.53% in the diet.

Effects on developmental toxicity

Description of key information

Key study. Test method similar to OECD Guideline 414. In a pre-natal developmental toxicity performed on rats no evidence of teratogenic effects was observed after oral administration at levels up to 1000 mg DUP/kg bw/day.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
not specified
Remarks:
(this information was not provided)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories (Saint-Germain-sur-l’Arbresle, France)
- Weight at study initiation: ca. 230 g average
- Housing: Mated females were singly housed in clear polycarbonate cages with stainless steel wire lids and virgin loose pulp as bedding (Alpha-Dri®, Dietex, Saint Gratien, France).
- Diet (e.g. ad libitum): Food pellets (UAR Alimentation, Villemoisson, France), ad libitum.
- Water (e.g. ad libitum): filtered tap water, ad libitum.
- Acclimation period: 1-2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 55 ± 15 %
- Photoperiod (hrs dark / hrs light): 12 light / 12 hours dark

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations were prepared weekly and stored in a dark place at room temperature. Stability was established for up to 2 weeks by gas chromatography analysis.

VEHICLE
- Concentration in vehicle: 0, 50, 100, 200 mg/mL, giving dose levels of 0, 250, 500, 1000 mg/kg/day.
- Amount of vehicle (if gavage): 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability was established for up to 2 weeks by gas chromatography analysis.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused: primiparous female (180–200 g) rats were housed overnight with adult males from the same strain and supplier.
- M/F ratio per cage: not specified
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: Sperm in vaginal smear referred to as day 0 of gestation.

Duration of treatment / exposure:
Days 6 - 20 of gestation
Frequency of treatment:
Daily
Duration of test:
From day 0 to day 21 of gestation, when dams were euthanized.
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
21-22
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dosage levels of the definitive study were based on the findings of a dose-range finding study in which pregnant rats (9 or 10/group) were given 0, 0.5 or 1 g/kg/day of DUDP by gavage, on GD 6 to GD 20.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Maternal body weights were recorded on GD 0, 6, 9, 12, 15, 18, and 21.

FOOD CONSUMPTION: Yes, food consumption recordings were made at 3 day intervals commencing on GD 6.

POST-MORTEM EXAMINATIONS: No data other than for uterine contents.

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early and late resorptions: Yes
- Number of distribution of foetuses in each uterine horn: yes
Fetal examinations:
- External examinations: Yes - all per litter
- Soft tissue examinations: Yes - half per litter
- Skeletal examinations: Yes - half per litter
- Head examinations: Yes - half per litter (as part of soft tissue examination)
- Other: Anogenital distance (AGD) was measured using a dissecting microscope with a micrometer eyepiece. The degree of trans-abdominal testicular migration (TTM) was determined by measuring the distance from the bladder neck to the lower pole of the testes using a dissecting microscope with a micrometer eyepiece.

Statistics:
The litter was used as the basis for the analysis of foetal variables.

Maternal body weights, body weight gain and food consumption of pregnant rats, number of corpora lutea, number of implantation sites and live foetuses, litter mean foetal body weight and AGD were analysed by one-way analysis of variance, followed by Dunnett’s test if differences were found.

The mean percentage of post-implantation loss, dead foetuses, resorptions, sex ratio (male foetuses per litter), and the proportion of affected foetuses per litter (calculated for each alteration) were evaluated by using the Kruskal–Wallis test, followed by the Mann–Whitney test if differences were indicated.

The rate of pregnancy and the number of litters with dead foetuses, resorptions, or foetal alterations were analysed by using Fisher’s test. Additional statistical evaluations were performed using the number of live foetuses per litter as a covariate for foetal body weight. Foetal AGD was also analysed with foetal body weight as covariant, using a linear mixed-effects model with two levels of variance in which litter effect was modelled with a nested random factor and dose and foetal weights as fixed factors. Least-squares analysis was carried out where applicable.

Treated groups were compared to their respective vehicle control.

All tests were reported at the 5% or 1% level of significance.

Clinical signs:
no effects observed
Description (incidence and severity):
No treatment-related clinical signs were noted.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
All pregnant animals survived to scheduled euthanization on GD 21.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no statistically significant changes in mean maternal body weights and body weight gains throughout the study.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Maternal food consumption was unaffected by treatment.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
Treatment did not influence drinking-water consumption.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Number of abortions:
no effects observed
Description (incidence and severity):
No abortions were registered either in treated groups or in the control group.
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
No significant differences were observed in the number of corpora lutea or incidence of pre-implantation loss. The number of implants was slightly but significantly lower than the concurrent control at the low and mid doses. There were no effects of DUDP on post-implantation loss.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There were no effects of DUDP on resorptions.
Early or late resorptions:
no effects observed
Description (incidence and severity):
There were no effects of DUDP on resorptions.
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no effects of DUDP on live fetuses.
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
No further influence on the prenatal development was detected.
Key result
Dose descriptor:
LOEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: decrease in implantation sites
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal general toxicity
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
There were no effects of DUDP on fetal body weights.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There were no effects of DUDP on live fetuses.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There were no effects of DUDP on fetal sex ratio (percent male fetuses per litter).
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Malformations occurred in one fetus at 0.25 g/kg/day (omphalocele) and in one fetus at 1 g/kg/day (diaphragmatic hernia). These isolated cases were considered incidental. Common external (i.e. club foot) variations were seen in single or few fetuses, with no indication of any adverse effects related to the administration of DUDP.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
An increased occurrence of lumbar ribs was observed in fetuses from the 0.5 and 1 g/kg/day dose groups, compared to control. The mean percentage of affected fetuses per litter amounted to 10.3, 20.8, 46.6, and 25.4 at 0, 0.25, 0.5, and 1 g/kg/day, respectively. The historical control range was from 6.8% to 19.4%. The incidence of affected litters was also significantly increased at the mid dose.

Long supernumerary ribs were only observed in one fetus at 0.25 g/kg/day and in one fetus at 1 g/kg/day. Otherwise, supernumerary 14th ribs were pinpoint ossification sites (78–88%) or were short, in the control and treated groups. There were no significant changes in the incidences of any other skeletal variations or in the ossification of metacarpals, metatarsals, and phalanges. The elevated number of ossified caudal vertebral centra in the DUDP-treated groups compared to control was not considered toxicologically meaningful. All fetuses had 26 presacral vertebrae.

Although the increase in the incidence of fetuses with short supernumerary lumbar ribs at 0.5 and 1 g DUDP/kg/day did not occur in a clear dose-related manner a relationship to treatment cannot be ruled out, and also the authors state that supernumerary lumbar ribs, especially longer ribs, could be regarded as being an indicator of changes in axial skeletal development. However, rudimentary supernumerary ribs are usually considered as common reversible variants in rodent bioassays and there were no other notable skeletal findings at any dose investigated. Thus, it is considered that no adverse effects were found up to the highest dose tested.

Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Common visceral (e.g. left umbilical artery) variations were seen in single or few fetuses, with no indication of any adverse effects related to the administration of DUDP.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Male AGD was identical to control at 0.25 g/kg/day, and it was slightly, although not significantly, reduced at 0.5 and 1 g/kg/day (3–4%). A statistically significant difference was only noted at 0.5 g/kg, after adjustment with the cubic root of fetal weight or when fetal body weight was used as covariate (with litter based analysis or mixed-effects model).

There was no male with mal-positioned testis and the pattern of trans-abdominal testicular migration was comparable across groups.
Key result
Dose descriptor:
LOEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
skeletal malformations
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effect observed at the highest dose tested
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Table 1. Maternal findings.

 

DUDP (g/kg/day)

 

0

0.25

0.50

1.00

No. dead/treated 

0/22

0/21

0/21

0/22

No. (%) pregnant

22 (100.0)

21(100.0)

21(100.0)

20(90.9)

Body weight (g)

 

 

 

 

GD 0 

230±13a

229±11

229±13

231±13

GD 6 

259±11

259±11

258±15

257±15

GD 9 

272±11

273±12

274±18

269±15

GD 12 

290±12

290±14

291±21

286±17

GD 15 

312±15

311±14

314±24

309±18

GD 18 

354±19

352±16

353±33

352±20

GD 21 

414±25

407±25

405±47

408±27

Gravid uterine weight (g)

107±13

98±23

96±29

103±15

Net body weight (g)b

307±16

309±17

309±25

305±20

Body weight change (g)

 

 

 

 

GD 0–6 

29±6

30±6

29±8

26±6

GD 6–9 

14±3

14±5

16±4

11±5

GD 9–12 

18±5

17±5

17±8

17±4

GD 12–15 

22±5

21±6

23±9

24±4

GD 15–18 

42±6

42±8

39±13

43±7

GD 18–21 

60±9

55±11

52±16

56±11

GD 0–21 

184±19

178±22

177±43

177±21

Net weight gain (g)c

78±14

80±12

81±23

74±16

Food consumption (g/day)

 

 

 

 

GD 0–6 

21±1

21±2

21±2

21±2

GD 6–9 

19±2

21±2

21±3

21±4

GD 9–12 

21±2

23±2

23±3

22±3

GD 12–15 

22±2

23±2

24±4

23±2

GD 15–18 

24±2

26±2

26±4

27±2

GD 18–21 

25±3

25±3

25±5

26±3

GD 0–21 

22±1

23±2

23±3

23±2

Body weight on GD 21 (b) or body weight gain during GD 0–21 (c) minus gravid uterine weight.

a Values are expressed as means±SD.

* Significant differences from the vehicle control, p < 0.05.

** Significant differences from the vehicle control, p < 0.01.

Table 2. Reproductive findings.

 

DUDP (g/kg/day)

 

0

0.25

0.50

1.00

All litters a  

22

21

21

20

No. corpora lutea 

15.7±1.5b

14.7±2.0

14.9±1.8

15.3±1.7

%Pre-implantation loss per litter

3.0±4.8

10.4±17.7

8.4±13.7

5.9±10.2

No. implantation sites per litter

15.2±1.8

13.2±3.3*

13.4±2.9*

14.3±1.7

%Post-implantation loss per litter c

6.4±10.8

2.5±5.9

8.0±21.4

5.6±5.7

No. litters with dead fetus

1

1

1

0

%Dead fetuses per litter

0.3±1.2

0.4±1.8

0.3±1.5

0.0±0.0

No. litters with resorptions

12

5

9

6

%Resorptions per litter 

6.1±10.7

2.2±4.4

7.7±21.5

3.7±7.2

Live littersd  

22

21

20

20

No. live fetuses per litter

14.2±1.9

12.9±3.3

13.2±2.7

13.8±2.0

%Male fetuses per litter

48.4±12.1

51.1±19.5

42.9±13.7

49.9±15.8

Fetal body weight (g)

 

 

 

 

All fetuses  

5.52±0.31

5.58±0.36

5.60±0.21

5.53±0.27

Male fetuses  

5.69±0.31

5.75±0.34

5.78±0.25

5.69±0.30

Female fetuses  

5.37±0.32

5.37±0.39

5.48±0.19

5.39±0.28

AGD (mm)  

 

 

 

 

No. litters  

18

17

16

16

Male fetuses  

2.96±0.12

2.95±0.15

2.85±0.11

2.86±0.15

Female fetuses  

1.04±0.06

1.06±0.06

1.07±0.06

1.09±0.06

AGD/body weight 1/3  

 

 

 

 

Male fetuses  

1.65±0.08

1.65±0.08

1.59±0.05*

1.60±0.09

Female fetuses  

0.59±0.03

0.60±0.03

0.61±0.03

0.62±0.04

a Includes all pregnant females at euthanization.

b Values are expressed as means±SD.

c [(No. resorptions plus dead fetuses)/No. Implantations]×100.

d Includes all animals with live fetuses at euthanization.

* Denotes significant differences from the vehicle control, p < 0.05.

Table 3. Fetal malformations and variations

 

Dose (g/kg/day)

 

0

0.25

0.50

1.00

No. fetuses (litters) examined a

 

 

 

 

External

312 (22)

270 (21)

264 (20)

275 (20)

Visceral

156 (22)

135 (21)

132 (20)

138 (20)

Skeletal

156 (22)

135 (21)

132 (20)

137 (20)

Malformations

 

 

 

 

Omphalocele

0

1 (1)

0

0

Diaphragmatic hernia

0

0

0

1 (1)

External variations

 

 

 

 

Club foot (unilateral)

0

0

1 (1)

0

Visceral variations

 

 

 

 

Umbilical artery, left

4 (4)

2 (2)

4 (4)

4 (3)

Distended ureter

1 (1)

0 (0)

0 (0)

0 (0)

Skeletal variations

 

 

 

 

Sternebra ossification

 

 

 

 

Dumbell, bipartite, incomplete or absent (5th)

2 (2)

3 (2)

2 (2)

0

Misaligned

1 (1)

0

0

0

Cervical rib(s)

2 (1)

3 (3)

2 (1)

4 (3)

14th rib(s), supernumerary (any)

17 (10)

29 (13)

60 ##(17 )*

32 # (12)

Long b

0

0

1 (1)

1 (1)

Thoracic vertebral centra, ossification

 

 

 

 

Bipartite, dumbbell and/or incomplete (one or two)

13 (8)

16 (11)

20 (10)

16 (7)

Lumbar vertebral centra, ossification, bipartite or incomplete

0

0

1 (1)

1 (1)

No. ossification centres

 

 

 

 

Metacarpals

4.00 ± 0.00 c

3.99 ± 0.05

4.00 ± 0.00

4.00 ± 0.00

Forelimb proximal phalanges

3.79 ± 0.33

3.67 ± 0.67

3.74 ± 0.29

3.81 ± 0.36

Metatarsals

4.95 ± 0.08

4.96 ± 0.16

4.99 ± 0.04

5.00 ± 0.01

Hindlimb proximal phalanges

2.26 ± 0.95

2.33 ± 1.12

2.15 ± 0.92

2.38 ± 1.19

Caudal vertebral centra

6.12 ± 0.37

6.59 ± 0.70 §

6.70 ± 0.57 §§

6.67 ± 0.65 §§

a The incidence of individual defect is presented as number of fetuses (number of litters).

b More than one third of the length of the preceeding rib

c Mean±SD.

* Denotes significant differences from control, p < 0.05 (Fisher’s test).

# Denotes significant differences from control, p < 0.05, (Mann–Whitney test).

## Denotes significant differences from control, p < 0.01, (Mann–Whitney test).

§ Denotes significant differences from control, p < 0.05, (Dunnett’s test).

§§ Denotes significant differences from control, p < 0.01 (Dunnett’s test).

Conclusions:
In a pre-natal developmental toxicity on diundecyl phthalate (DUP) no evidence of teratogenic effects was observed after oral administration at levels up to 1000 mg/kg bw/day.

Executive summary:

A pre-natal developmental toxicity test was performed with DUP following a method similar to OECD Guideline 414. Groups of 21-22 pregnant Sprague-Dawley rats were administered 0, 250, 500, or 1000 mg/kg/day of DUDP diluted in olive oil by gavage on gestation days 6–20. DUDP had no adverse effects on maternal body weight and food consumption. The number of live fetuses, percent of post-implantation loss and resorptions, fetal sex, and fetal body weights were not affected. A statistically significant decrease in implantation sites was observed at doses of 250 and 500 mg/kg/day. The significance of this observation was considered unclear and authors suggest that female fertility and reproduction need to be further investigated. Small decreases in the anogenital distance of male fetuses were noted at 500 and 1000 mg/kg/day. This finding provides limited evidence that DUDP might have marked effects on the male sexual differentiation and thus further studies are needed to be conducted to evaluate the effects of an in utero exposure on the male sexual development in the fetal, young, and adult offspring. No evidence of teratogenic effects was observed after oral administration at levels up to 1000 mg/kg bw/day. There were no significant changes in the incidence of external, visceral or skeletal variations, except for an increase in the incidence of foetuses with short supernumerary lumbar ribs at 500 and 1000 mg/kg bw/day. Although this variation did not occur in a clear dose-related manner, a relationship to treatment cannot be ruled out and the authors state that supernumerary lumbar ribs, especially longer ribs, could be regarded as being an indicator of changes in axial skeletal development. However, rudimentary supernumerary ribs are usually considered as common reversible variants in rodent bioassays and there were no other notable skeletal findings at any dose investigated. The NOAEL is therefore regarded as being 1000 mg/kg bw/day, the highest dose investigated.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The key study has a Klimisch score = 2
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Key study: A pre-natal developmental toxicity test was performed with DUDP following a method similar to OECD Guideline 414. Groups of 21-22 pregnant Sprague-Dawley rats were administered 0, 250, 500, or 1000 mg/kg/day of DUDP diluted in olive oil by gavage on gestation days 6–20. DUDP had no adverse effects on maternal body weight and food consumption. The number of live fetuses, percent of post-implantation loss and resorptions, fetal sex, and fetal body weights were not affected. A statistically significant decrease in implantation sites was observed at doses of 250 and 500 mg/kg/day. The significance of this observation was considered unclear and authors suggest that female fertility and reproduction need to be further investigated. Small decreases in the anogenital distance of male fetuses were noted at 500 and 1000 mg/kg/day. This finding provides limited evidence that DUDP might have marked effects on the male sexual differentiation and thus further studies are needed to be conducted to evaluate the effects of an in utero exposure on the male sexual development in the fetal, young, and adult offspring. No evidence of teratogenic effects was observed after oral administration at levels up to 1000 mg/kg bw/day. There were no significant changes in the incidence of external, visceral or skeletal variations, except for an increase in the incidence of foetuses with short supernumerary lumbar ribs at 500 and 1000 mg/kg bw/day. Although this variation did not occur in a clear dose-related manner, a relationship to treatment cannot be ruled out and the authors state that supernumerary lumbar ribs, especially longer ribs, could be regarded as being an indicator of changes in axial skeletal development. However, rudimentary supernumerary ribs are usually considered as common reversible variants in rodent bioassays and there were no other notable skeletal findings at any dose investigated. The NOAEL is therefore regarded as being 1000 mg/kg bw/day, the highest dose investigated.

Justification for classification or non-classification

Based on the available information, the substance is not classified for toxicity to reproduction in accordance with CLP Regulation (EC) no 1272/2008.

Additional information